Australopithecus species at Sterkfontein Member 4, South Africa (2.07–2.61 Ma), is highly contentious, and quantitative assessments of craniodental and postcranial variability remain inconclusive. Using geometric morphometrics, we compared the sacrum of the small-bodied, presumed female subadult Australopithecus africanus skeleton Sts 14 to the large, alleged male adult StW 431 against a geographically diverse sample of modern humans, and two species of Pan, Gorilla, and Pongo. The probabilities of sampling morphologies as distinct as Sts 14 and StW 431 from a single species ranged from 1.3 to 2.5% for the human sample, and from 0.0 to 4.5% for the great apes, depending on the species and the analysis. Sexual dimorphism and developmental or geologic age could not adequately explain the differences between StW 431 and Sts 14, suggesting that they are unlikely to be conspecific. This supports earlier claims of taxonomic heterogeneity at Sterkfontein Member 4.The presence of multiple  Australopithecus africanus specimens from Sterkfontein Member 4, South Africa. By comparing the two fossil hominins to a diverse sample of modern humans and great apes, the authors conclude that the observed morphological differences are unlikely to occur within a single species, supporting earlier claims of taxonomic heterogeneity within the Australopithecus africanus hypodigm.Fornai et al. present a geometric morphometric study of the sacrum in two alleged  Australopithecus africanus, although several authors noted the remarkably high morphological variability within these assemblages11. This led Clarke12 to propose the presence of a second Australopithecus species at Sterkfontein Member 4, which he attributed to A. prometheus, a species name originally given to fossils from Makapansgat13. Recent studies of the StW 573 “Little foot” skeleton from Sterkfontein Member 2 renewed debates on the functional biology and taxonomy of the Plio-Pleistocene hominins from South Africa16. Nevertheless, the presence of two closely related Australopithecus taxa at Sterkfontein Member 4 is not widely accepted18 because of conflicting interpretations and the fragmentary preservation of the fossils. Moreover, quantitative studies focused mainly on the craniodental remains and rarely considered morphological variation within a broad comparative setting.Traditionally, all early hominin fossils from Taung, Sterkfontein Member 4 and Makapansgat have been attributed to 22 and StW 43127 are the best-preserved, albeit incomplete skeletons from Sterkfontein Member 4. In Sts 14, the unfused apophyses of the iliac crests and ischial tuberosities and the partially fused epiphyseal plates of the sacral alae suggest an age of about 15 years based on modern human standards29. Its acetabulum, which already reached adult dimensions, predicts a body size of 25.4 kg30, and the pubic morphology suggests a female sex24. StW 431, an adult individual that probably weighed considerably more than 40 kg31, was possibly of male sex based on its greater robusticity and larger body size. Both Sts 14 and StW 431 are conventionally attributed to A. africanus. However, the StW 431 sacrum is narrower and more elongated while Sts 14 is comparatively gracile and wide relative to its small sacral body32 , which lacks well-developed upper lateral angles of the transverse processes33.Sts 14n = 74) and an extensive sample of extant great apes . As Sts 14 and StW 431 do not patently show sacral segmentation anomalies, we confine our comparative sample to specimens without lumbosacral transitional vertebrae. We apply two different morphometric approaches, one in which we register the landmark configurations by a Generalized Procrustes Analysis (GPA) of all 113 landmarks, and one in which all landmarks are registered based on a GPA of the 13 landmarks on the body of the first sacral vertebra only (Procrustes fit on a subset of landmarks35). This second geometric morphometric analysis is chosen because the relative size and shape of the sacral alae vary greatly with respect to the sacral body. The datasets originating from the GPA are analysed both in shape space and form space. To assess the morphological heterogeneity between the StW 431 and Sts 14 sacra, their Procrustes distance is compared to all pairwise differences within our extant species sample. Our investigation reveals that the morphological differences between Sts 14 and StW 431 could not be fully explained by sexual dimorphism or differing developmental or geologic age, suggesting that taxonomic heterogeneity may contribute to the morphological variability within Sterkfontein Member 4 Australopithecus.Here, we quantify the morphological differences of the sacrum between Sts 14 and StW 431 using a comparative approach. We explore how the magnitude of their shape differences compares to within-species variation of modern humans and extant great ape species while taking into account factors such as sexual dimorphism and differences in individual and geologic age. Accordingly, we use geometric morphometrics based on 113 3D landmarks  of the shape coordinates based on the GPA of all landmarks, Sts 14 and StW 431 plotted at the opposite sides of the modern human distribution, whereas all extant great apes were clearly separated from modern humans along PC1  . Size-related shape variation accounted for 12.3% of total sacral shape variation within the full sample but for only 3.3% within the hominin sample . The Procrustes distance, a measure of overall shape difference, between StW 431 and Sts 14 was among the largest observed distances within the modern human sample, including both adults and subadults  and, hence, are unlikely to account for the differences in sacral shape between StW 431 and Sts 14. It is also unlikely that the shape differences between the fossils result from differences in individual age because in our PCA analysis 41 which in turn might have come from the vicinity of the flowstone topping Sterkfontein Member 438. However, flowstones are often post-depositional infillings of voids within the cave sediments and thus only provide a minimum age for the fossils42. On the other hand, almost all fragments of the StW 431 skeleton were found in two adjacent square yards at a mean depth of 7 m below datum, while many other Member 4 fossils were recovered from deeper deposits. Parts of the StW 431 skeleton were vertically distributed between a depth of 6.5 m below datum (most of the vertebrae and right arm) and a depth of 9.0 m (right pubis fragment)43. This distribution reflects a complex taphonomy of the StW 431 skeleton, which might be explained by disturbance of a talus cone by later collapses45. Thus, although the dating of the StW 431 remains is challenging, there is no indication that they are markedly different in age from Sts 14.According to the most recent U-Pb dating of flowstones46. Moreover, P. pygmaeus and P. abelii diverged about 400 ka years ago47 but were indistinguishable in the PCA. Even Pan troglodytes and P. paniscus were comparable in sacrum shape although they diverged 1.6 Ma ago, with extensive gene flow until at least 200 ka ago48. Gorilla gorilla and G. beringei diverged 1.75 Ma ago, with substantial gene flow until at least 20 ka ago49, but also largely overlapped in sacrum shape. Thus, sacrum shape appears to be evolutionarily conserved, presumably due to stabilizing selection imposed by biomechanical and obstetric demands50.In any case, a possible difference in geological age between StW 431 and Sts 14 can only partially, if at all, account for the differences in sacrum shape. The human populations in our sample completely overlapped in the PCA of sacrum shape, even though some of them have an evolutionary depth of about 260–350 ka51, such border shifts are frequent in hominin fossils and are often associated with border shifts at the lumbosacral junction52.Since Sts 14 and StW 431 preserve only the first two and a half sacral vertebrae, it is impossible to completely rule out transitional vertebrae in either of these specimens, hence this aspect should be investigated further. In fact, Sts 14 shows a segmentation anomaly at the thoracolumbar transitionA. africanus sacrum was as variable in shape as that of modern humans and extant great ape species, it is unlikely though possible that Sts 14 and StW 431 belong to a single species, thereby supporting earlier claims of taxonomic heterogeneity at Sterkfontein Member 4. Yet, as neither Sts 14 nor StW 431 is associated with craniodental remains, it remains impossible to infer which one of these partial skeletons, if any, belongs to A. africanus.In conclusion, our results show that, under the assumption that the A. afarensis). Our geographically diverse modern human sample comprised 63 individuals including Central Europeans (n = 28), Western Africans (n = 13), Khoe-Sān (n = 7) and Pygmies (n = 2), Indians (n = 8), and Fuegians (n = 5). The great ape sample included P. paniscus (n = 8), P. troglodytes (n = 22), G. beringei (n = 10), G. gorilla (n = 23), P. abelii (n = 7), and P. pygmaeus (n = 24)  with a developmental age similar to that of Sts 14 (16–17 years). Specimens with damaged or eroded surface or with patent asymmetry were excluded, as well as individuals with lumbosacral transitional vertebrae. 3D-surface models of the sacra were obtained using a high-resolution optical 3D-surface scanner 55. In instances where CT data were available, the meshes were generated by segmentation using the software Amira (www.fei.com). The skeletal collections and the source of the surface models are listed in Table We compared the partial sacrum of StW 431 and Sts 14 to a sample of 157 sacra from adult modern humans and great apes of known sex, as well as the complete sacrum of the presumed female A.L. 288-1 58. The landmarks were collected in Viewbox 4 , and analysed in Evan Toolbox (www.evan-society.org). The sliding of the semilandmarks was based on the minimum bending energy criterion after relaxation against the consensus shape. The geometric morphometric analysis after Procrustes fit based on a subset of 13 landmarks on the body of the first sacral vertebra was performed in the R software environment59 using a code written explicitly for this purpose. The software PAST was used for generating the PCA plots.Standard geometric morphometric analyses were performed using principal component analysis (PCA) of the Procrustes shape coordinates both in shape space and form space, the latter by augmenting the shape coordinates with the natural logarithm of centroid size (lnCS)59  after the GPA analysis in both shape and form space, and after a Procrustes fit based on the subset of 13 landmarks on the body of the first sacral vertebra. The percentages of Procrustes distances higher than that observed between StW 431 and Sts 14 were computed for all pairwise comparisons and separately also for male–female pairs only, both on the genus and species level, if at least 20 individuals could be included. An intra- and interobserver error assessment was performed by C.F. and V.A.K. confirming high precision of the landmark configurations . Variation in sacral size was assessed by box plots of lnCS in the R software environmentFurther information on experimental design is available in the Description of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary InformationSupplementary Data 1Supplementary Data 2Reporting SummaryPeer Review File


Dengue fever (DF) is a viral infection that is common in tropical countries and represents a significant cause of global morbidity and mortality. Despite its prevalence and severity, treatment options for DF remain limited and consist primarily of supportive measures. Several recent studies have concluded that micronutrient supplementation may improve clinical outcomes in patients with DF, but no review has summarized and synthesized these findings. We conducted a literature review to identify articles investigating the effect of micronutrient supplementation on clinical outcomes among patients with DF. We found several studies which indicated that supplemental vitamin C, vitamin D, vitamin E, and zinc may be useful adjuncts in DF treatment. Folic acid supplementation did not appear to affect clinical outcomes. The reviewed studies have significant limitations including small sample sizes and limited data about the baseline nutritional status of study subjects. We identify a need for additional high-quality randomized trials to elucidate the role of micronutrient supplementation in DF treatment. Dengue hemorrhagic fever can ultimately lead to dengue shock syndrome, a severe condition consisting of circulatory collapse and sometimes death.3 The pathophysiology of these syndromes is complex, but dengue hemorrhagic fever and dengue shock syndrome are thought to be caused in part by an excessive cytokine response leading to increased vascular permeability, which contributes to vascular leakage and subsequent shock.4 Growing evidence also suggests that oxidative stress may play a role in DF pathogenesis.5Dengue fever (DF) is a mosquito-borne viral infection that is common in tropical and subtropical areas worldwide.9 Treatment options for DF patients remain limited, as no effective antiviral drugs are currently available.10 Current guidelines for clinical treatment of DF patients largely focus on supportive measures, with specific attention paid to management of fluid status and hemodynamic parameters among those patients who develop dengue hemorrhagic fever and dengue shock syndrome.12Dengue fever and its complications are a significant cause of global morbidity and mortality, with approximately 300 million infections and 10,000 deaths per year.15 as these supplements are generally inexpensive and some may offer hypothetical benefits to dengue patients via numerous potential mechanisms, including modulation of the host immune response20 and antioxidant effects.21 Proposed mechanisms of clinical benefit in DF for several micronutrients are outlined in Providers caring for DF patients sometimes provide micronutrient supplements as an adjunct to standard supportive care,22 vitamin D,23 vitamin E,24 folic acid,14 and zinc.25 No prior literature review has summarized the findings of these clinical studies. Our goals in this review, therefore, were to consolidate and synthesize the findings of studies between 2010 and 2020, which investigated the role of micronutrient supplementation in the clinical care of patients with DF, and to identify gaps in the current literature to guide future research on this topic.Several recent studies have sought to evaluate the efficacy of micronutrient supplementation among DF patients. These studies have investigated associations between clinical outcomes and supplementation of various micronutrients, including vitamin C,Supplemental Appendix 1.We conducted a literature search using five major databases: MEDLINE, Global Health, Scopus, Academic Search Complete, and Embase. Search terms including “dengue” and “breakbone fever” were paired with micronutrient-related terms such as “vitamin” and “supplement” and the names of specific micronutrients, including “zinc,” “folate,” and “iron.” We restricted our search to human studies, English language articles, and articles published between January 1, 2010 and March 23, 2020. All abstracts and articles were managed through EndNote X9 . We initially de-duplicated records using the “Find Duplicates” feature in EndNote X9, and then screened each title for terms related to micronutrients and DF. We subsequently reviewed each abstract with possible relevance and excluded those in which micronutrient supplementation in human DF patients was not discussed. We then performed full-text review of each included article and removed those which did not contain novel findings relevant to our research question. Our complete search strategy is attached as Aedes aegypti mosquitoes, articles commenting on the use of traditional herbal remedies to manage DF, and research investigating the impact of micronutrients on dengue-infected cells in vitro. We completed full-text review of the remaining eight records. One additional record was then excluded, as it was a letter to the editor that did not present any novel findings relevant to our research question. The remaining seven studies  were included in our review . Study features that resulted in lower scores included failure to adequately address possible confounders, inadequate provision of information regarding withdrawals and dropouts, and lower-quality study types .Of the seven reviewed studies, six were assessed using the Quality Assessment Tool for Quantitative Studies .13 and Rammohan et al.22 both investigated the effect of supplemental vitamin C in the management of DF. Ramalingam et al.13 conducted a retrospective study of 200 patients infected with dengue virus across various hospitals in India. The authors stated that 35.5% of patients were aged 0–15 years, but did not provide any other information regarding the ages of study participants. One hundred of these patients were given supplemental vitamin C and 100 were not, but information about dosing was not provided. Among the patients receiving vitamin C, 67% received oral vitamin C, 28% received intravenous (IV) vitamin C, and 5% (described by the authors as “more severe cases”) received both. The authors determined that the average increase in platelet count was greater among patients who received vitamin C than among those who did not . Patients receiving vitamin C were also hospitalized significantly fewer days than those who did not receive vitamin C. Of patients who received vitamin C, 39% had hospitalizations lasting fewer than 5 days. Of those who did not receive vitamin C, only 3% had hospitalizations lasting fewer than 5 days. In describing outcomes, the authors did not differentiate between patients who received oral vitamin C and patients who received IV vitamin C. Of note, this study did not include any information about patients’ baseline nutritional status, did not state whether patients were deficient in vitamin C, nor did it contain any information about the patients’ diets.Ramalingam et al.22 conducted a prospective observational study of 123 DF patients with thrombocytopenia at an academic medical center in Bangalore, India. The results were summarized in a conference abstract, which did not provide effect sizes for the study’s results or any demographic information about the enrolled patients. Study subjects were randomized into two groups: patients in one group received standard care with no vitamin C (n = 60), whereas patients in the other group received standard care in addition to 500 mg of oral vitamin C four times/day (n = 63). The authors assessed hemoglobin and white blood cell (WBC) counts at day 7 of treatment. They found no change in hemoglobin in either group, and noted that WBC counts increased in both groups. White blood cell counts in the treatment group, however, increased significantly more than those in the nontreatment group (P = 0.00).Rammohan et al.23 conducted an RCT of 124 patients suffering from DF at an academic medical center in Rawalpindi, Pakistan. The mean age of the study subjects was 33 years, but baseline data were not provided separately for the intervention and control groups. Patients were randomized either to receive a single dose of 200,000 international units of vitamin D (n = 62) in addition to standard care, or to receive standard care only (n = 62). Both groups were followed for development of dengue hemorrhagic fever or dengue shock syndrome. Among patients who received the vitamin D supplement, only one patient (1.6%) developed dengue hemorrhagic fever. In the group that did not receive the supplement, 17 patients (27%) developed dengue hemorrhagic fever. Those not receiving vitamin D were more likely to develop dengue hemorrhagic fever . The relative risk of progressing to dengue hemorrhagic fever in patients who received vitamin D compared with those who did not receive vitamin D was 0.0588 . However, this study did not assess whether patients were vitamin D deficient at baseline, and did not contain any information about the patients’ diets.Zaman et al.21 conducted an RCT of 127 children aged 5–12 years with DF at a tertiary care hospital in Colombo, Sri Lanka. They were randomized to one of two groups: 1) the experimental group, in which patients received either 200 mg of vitamin E once daily for 7 days (for children aged 5–9 years) or 400 mg of vitamin E once daily for 7 days (for children aged 10–12 years) in addition to supportive care; or 2) the control group, in which patients received only supportive care. A wide array of clinical, biochemical, and hematological parameters were measured across both groups including duration of capillary leaking, aspartate transaminase (AST), alanine transaminase (ALT), albumin, serum cholesterol, serum calcium, WBC count, platelet count, and packed cell volume (PCV). Each of these parameters was evaluated twice per day from day 2 to day 7 of hospitalization. The only statistically significant differences between the two groups were as follows: the group receiving vitamin E had a shorter mean duration of capillary leakage , a higher mean WBC count on day 6 , a higher mean albumin on days 2.5  and 5, a lower mean PCV at days 3  and 3.5 , a lower mean ALT level on days 4  and 5 , and a lower mean AST level on days 3 , 4 , and 5 . There was no difference in the 1) duration of hospital stay or 2) occurrence of capillary leakage. This study did not assess patients’ baseline nutritional status and did not evaluate for vitamin E deficiency at baseline.Chathurangana et al.24 performed a prospective randomized open-blinded evaluation of 59 patients with DF at a tertiary teaching hospital in Lucknow, India. The intervention group included 31 DF patients who received 400 mg vitamin E once daily in addition to standard care, whereas the control group consisted of 28 patients who received standard care without vitamin E. Most study subjects were male, and the mean age of enrolled patients was 28.7 years in the intervention group and 31.7 years in the control group, with no statistically significant difference in mean age between the two groups. Platelet counts in both groups were measured on days 0, 1, 4, and 7 of illness. The authors found that mean platelet counts among those patients in the intervention group were significantly higher than those among patients in the control group on days 4  and 7  of treatment. Of note, this study did not provide information about patients’ baseline nutritional status, did not state whether they were vitamin E deficient at baseline, and did not assess whether patients experienced side effects.Vaish et al.14 conducted a retrospective observational study of 1,464 patients who were hospitalized with DF at an urban academic medical center in Karachi, Pakistan. Patients had a mean age of 36.5 years. The study’s primary goal was to determine whether there was a significant difference in the duration of recovery of thrombocytopenia between patients who received folic acid supplements and patients who did not. As secondary endpoints, they also investigated whether any differences existed in the mortality rate, likelihood of needing admission to the intensive care unit, length of stay, and creatinine levels between the two groups. Of the 1,464 patients, 1,322 (90.3%) received folic acid and 142 (9.7%) did not. There were no significant differences between the two groups with respect to any of the above endpoints, but the authors did not provide effect sizes. This study did not assess for baseline nutritional status, did not evaluate for zinc deficiency, and did not assess for side effects among patients receiving supplements.Syed et al.25 conducted an RCT in 50 children (mean age 6.3 years) with DF at an academic medical center in Bangkok, Thailand. Patients were randomized to receive either: 1) 15 mg oral bis-glycinate zinc three times daily for 5 days or until defervescence, or 2) placebo. Baseline zinc deficiency was present in 48% of patients in the treatment group and 44% of patients in the control group, with no statistically significant difference between the two groups. The authors found no statistically significant difference in mean time to defervescence between the supplement group and the placebo group . However, the mean duration of hospitalization was shorter in the supplementation group than in the placebo group .Rerksuppaphol and Rerksuppaphol27 Vitamin C supplementation was associated with higher platelet counts,13 shorter duration of hospitalization,13 and greater increases in WBC counts.22 Vitamin D supplementation was associated with lower likelihood of progression to dengue hemorrhagic fever.23 Vitamin E supplementation was associated with improvements in various clinical and hematological parameters,21 including significantly higher subsequent platelet counts among patients admitted for thrombocytopenia.24 Folic acid supplementation was not associated with a change in clinical outcomes.14 Zinc supplementation was associated with shorter duration of hospitalization.25In this review, we describe seven studies published between 2010 and 2020 which analyzed the association between micronutrient supplementation and clinical outcomes in patients with DF. We evaluated six of these studies using the Quality Assessment Tool for Quantitative Studies and found that they were of varying quality, with three receiving a global rating of “weak,” two receiving a global rating of “moderate,” and one receiving a global rating of “strong.” In all studies that provided dosing information, the doses used were between 2.63 times and 500 times higher than the daily recommended nutrient intake suggested by the WHO and the Food and Agriculture Organization of the United Nations.15 summarized existing work on micronutrients and dengue, but at the time of its publication, there were only two articles evaluating the use of micronutrient supplements in the clinical treatment of DF patients. These two articles were the 2012 Vaish et al.24 study described in our review and a 2009 case series by Sánchez-Valdéz et al.28 The Sánchez-Valdéz et al. case series concluded that supplementation of vitamin D3 and calcium may result in “improved overall clinical condition and shorter duration of symptoms of DF.”28 This assessment, however, was based on only five cases and did not have a control group. The other articles included in the Ahmed et al.15 review described laboratory studies of dengue virus–infected cells,30 observational studies assessing associations between vitamin D receptor polymorphisms and DF disease in humans,32 laboratory studies of dengue virus–infected mice,35 and noninterventional studies analyzing the serum or plasma components of DF patients.41 None of these studies directly investigated any clinical interventions for DF. Our review, performed 6 years after the Ahmed et al.15 review was published, identified six new clinical studies assessing micronutrient supplementation among DF patients, which may reflect a growing interest in this topic.There is limited previous research investigating relationships between micronutrient supplementation and clinical outcomes in DF patients. A 2014 review by Ahmed et al.24 and six did not state whether patients were deficient in the micronutrient to be supplemented.24 Five of the studies did not assess whether patients experienced any side effects related to the supplements.24 None of the seven studies contained any information about study subjects’ diets. Six studies had small sample sizes, with 200 or fewer total participants.25 One study did not provide any information about the micronutrient doses provided and pooled data from multiple hospitals using multiple routes of administration.13 One study investigated a wide array of laboratory parameters across many days and reported a handful of statistically significant results which may have limited clinical importance.21 Another study was described only in a conference abstract, which provided no effect sizes and contained very limited background information about the study population.22Although six of the seven articles in this review identified clinical benefits of supplementation of vitamin C, vitamin D, vitamin E, and zinc in DF patients, these findings must be interpreted in light of the studies’ significant limitations. Of the seven studies described, five did not include any information about patients’ baseline nutritional status,15 there is a need for additional high-quality studies of this topic. To obtain the most robust evidence possible, we recommend RCTs with large sample sizes and clinically meaningful endpoints. Studies should provide clear baseline data regarding both intervention and control groups with respect to possible confounders. Rates of withdrawals and dropouts should be stated explicitly, and authors should provide effect sizes in their results. We further recommend that future studies use consistent micronutrient doses, provide detailed demographic data about study subjects, and clearly specify the frequency, duration, and route of administration for each supplement administered. In addition, it is essential that future research investigates whether the benefits of micronutrient supplementation are seen in all patients or only in those with specific nutritional deficiencies. Therefore, future studies should provide baseline data about study subjects’ nutritional status, such as relevant micronutrient deficiencies, anthropometrics, laboratory tests such as prealbumin, or inventories of nutritional intake. Information about the diet provided to hospitalized patients should also be included. Furthermore, we agree with the recommendations of Ahmed et al.15 that future studies should include both adults and children, and should assess the impact of nutritional interventions on dengue of all levels of severity using standardized WHO clinical severity case definitions. These steps will help to provide clearer evidence of the potential benefits micronutrient supplementation may offer to DF patients.In this review of the literature, we found studies demonstrating possible improvement of DF clinical outcomes associated with the supplementation of vitamin C, vitamin D, vitamin E, and zinc. However, these studies have significant limitations, including small sample sizes and failure to assess baseline nutritional status. As micronutrient supplementation may represent a simple, low-cost adjunct to standard medical care,Supplemental materials


We conclude that the DDAH1 gene is associated with ADMA concentration and the incidence of DCI in SAH patients, suggesting a pathophysiological link between gene, biomarker, and clinical outcome in patients with SAH.Delayed cerebral ischemia (DCI) often causes poor long-term neurological outcome after subarachnoidal hemorrhage (SAH). Asymmetric dimethylarginine (ADMA) inhibits nitric oxide synthase (NOS) and is associated with DCI after SAH. We studied single nucleotide polymorphisms (SNPs) in the NOS3, DDAH1, DDAH2, PRMT1, and AGXT2 genes that are part of the L-arginine–ADMA–NO pathway, and their association with DCI. We measured L-arginine, ADMA and symmetric dimethylarginine (SDMA) in plasma and cerebrospinal fluid (CSF) of 51 SAH patients at admission; follow-up was until 30 days post-discharge. The primary outcome was the incidence of DCI, defined as new infarctions on cranial computed tomography, which occurred in 18 of 51 patients. Clinical scores did not significantly differ in patients with or without DCI. However, DCI patients had higher plasma ADMA and SDMA levels and higher CSF SDMA levels at admission. DDAH1 SNPs were associated with plasma ADMA, whilst AGXT2 SNPs were associated with plasma SDMA. Carriers of the minor allele of DDAH1 rs233112 had a significantly increased relative risk of DCI (Relative Risk = 2.61 (1.25–5.43),  Subarachnoidal hemorrhage (SAH) is a major neurosurgical emergency. Vasospasm and cerebral ischemia often occur with some delay during intensive care unit (ICU) treatment of SAH; delayed cerebral ischemia (DCI) is associated with poor clinical outcome . DespiteThe L-arginine–nitric oxide (NO) pathway is a major regulator of cerebral vascular tone . NO-mediBoth ADMA and SDMA are formed during posttranslational protein methylation by protein arginine N-methyltransferases (PRMTs) . In manyClinically, ADMA is associated with cardiovascular events and mortality ,17, whilGenotyping studies showed that single nucleotide polymorphisms (SNPs) in the NOS3 gene are associated with an increased incidence of DCI after SAH ,25 and wWe included 51 consecutive patients who had been admitted to the Intensive Care Unit of the University Medical Center Hamburg-Eppendorf with acute subarachnoidal hemorrhage. The aneurysmal pathogenesis was diagnosed using CT angiography or digital subtraction angiography; in 47 patients, an aneurysm was detected, and in four patients a perimesencephalic subarachnoidal hemorrhage was diagnosed. In 38 patients, external ventricular drain (EVD) insertion was performed on the day of admission because elevation of intracranial pressure was documented. The local ethics committee approved the study protocol , and written informed consent was obtained from all participants or their next of kin.2 35–45 mmHg) and normovolemia were controlled closely, and blood pressure was monitored continuously by invasive monitoring. Norepinephrine was primarily used for therapy of episodes of hypotension.The aneurysms were secured by intravascular coiling or surgical clipping as early as possible, at least within 24 h after admission. In eight cases, a non-interventional treatment approach was chosen. Subsequent adjunctive therapies and CT scans were administered when appropriate in adherence with international guidelines defined by the Neurocritical Care Society’s Multidisciplinary Consensus Conference and the American Heart Association (AHA) ,32. In aPatients were included on the day of bleeding and were reviewed on days three, six, eight, 12, and 15 after hemorrhage, including a standardized medical history, blood and CSF samples drawn from arterial catheters and external ventricular drains, respectively, and the Glasgow Coma Scale (GCS). CSF samples were only collected from patients with EVD inserted for clinical needs; for ethical reasons, no lumbar punctures were performed solely for study purposes.At admission, clinical assessment of patients was performed according to standardized procedures including the World Federation of Neurological Surgeons (WFNS) score , the GCSThe primary outcome parameter was DCI, which was assessed by cranial CT or magnetic resonance imaging performed when signs of neurological impairment became visible to identify new cerebral infarction according to current standards . TranscrAt discharge, neurological outcome was determined using the Glasgow Outcome Scale (GOS). Poor neurological outcome was defined as GOS < 4. In addition, outcome including the GOS was assessed at 3 months after discharge by patient visits or telephone interviews.We performed an extensive literature search using PubMed and the National Center for Biotechnology Information (NCBI) SNP database for publications linking SNPs in the DDAH1, DDAH2, ARG1, ARG2, AGXT2, NOS3, and PRMT1 genes to outcome parameters related to the pathophysiology of subarachnoidal hemorrhage, ischemic stroke, and vasospasm. These genes code for the key enzymes in the L-arginine–ADMA/SDMA–NO pathway  30]. Su. Su30]. DNA from blood samples was isolated using QIAamp DNA Blood Mini Kit according to the manufacturer’s protocol . DNA was eluted from the columns in a volume of 20 µL. To further purify and concentrate the isolated DNA for SNP genotyping, DNA was precipitated by ethanol precipitation. The DNA pellet was washed once and the resuspended in 20–100 µL H20. DNA concentration was determined using a nanophotometer NP60 .SNP genotyping was performed using single-tube human TaqMan SNP Genotyping Assays . Each reaction mix contained 10 ng template DNA, 0.5µL specific TaqMan SNP Genotyping Assay and 5 µL of 2xTaqPath ProAmp™ Mastermix in a total reaction volume of 10 µL. The polymerase chain reaction (PCR) was performed in a QuantStudio™ 5 Real-Time PCR System  using the following PCR program: pre-read of 30 s at 60 °C, enzyme activation at 95 °C for 5 min, 40 cycles of denaturation  and annealing , followed by a last post-read of 30 s at 60 °C. Allelic calls were identified by Quant Studio Design and Analysis Software .At baseline, non-fasting blood and CSF samples were drawn from arterial catheters (N = 51) and external ventricular drains , respectively. For ethical reasons, no patient received any interventional measures for study-related sample collection. Samples were centrifuged immediately and stored at −20 °C until analysis. ADMA, SDMA, and L-arginine were determined by liquid chromatography-tandem mass spectrometry (LC–MS/MS) using a previously validated method . In brie2 test. Haplotypes were constructed for the DDAH1 gene based upon the frequency of homozygosity for the major and minor alleles, respectively. The associations of DDAH1 haplotypes with outcome (DCI) were tested for significance by the Chi2 test. Data are presented as median with 25th and 75th percentiles, or as mean with standard error of the mean. For all tests, p < 0.05 was considered significant.All statistical analyses were performed using SPSS . All variables were tested for normal distribution using the Kolmogorov–Smirnov test. Differences between groups were tested for significance by using either the nonparametric Mann–Whitney U test for two groups or the Kruskal–Wallis analysis of variance for more than two groups. Correlations were calculated using linear regression or the Spearman test. Allele frequencies of the genes of interest (GOI) were compared to the European reference population or between groups using contingency tables and Fisher’s exact test. Biomarker levels between genotypes were tested for statistically significant differences by ANOVA followed by the Scheffé f-test, and associations of genotypes with outcome (DCI) were tested for significance by the Chip = 0.039), but no significant associations of EVD with the incidence of DCI , p = 0.286) nor vasospasm , p = 0.305).We included in this study 51 patients who were admitted to our Emergency Department with evidence for subarachnoidal hemorrhage. Patients had a mean age of 54.8 ± 13.1 years; 25% of them were men. A total of 43 patients were treated interventionally with either clipping or coiling of the aneurysmal location; 47 patients received nimodipine treatment. The Glasgow Coma Scale was 8.6 ± 5.6 on the day of admission, the WFNS score was 3.4 ± 1.7, and the Hunt and Hess index was 3.1 ± 1.4. Further demographic and anthropometric characteristics of the cohort are listed in p = 0.02; p = n.s.; p = n.s.), as well as a significantly higher rate of re-intervention . Vasospasms occurred slightly, but not significantly more frequently in patients with DCI (10 vs. 8 patients) than without DCI .A total of 18 patients developed DCI during the course of in-patient treatment. At admission, there were no significant differences in any of the demographic and anthropometric parameters between patients who later developed DCI or not . Initialp = 0.04) and SDMA at admission  were significantly higher in patients who later developed DCI than in those who did not. SDMA concentration was also significantly higher in CSF of patients with DCI than without . By comparison with the European reference population of the 1000 Genomes project, there were significant deviations from the expected distribution of alleles in four of the SNPs that we studied: For NOS3 rs891512, the major allele was more frequent in our cohort, whilst for the DDAH1 rs1241321 and DDAH1 rs480414 SNPs and the DDAH2 rs805304 SNP, the minor allele was more frequent in our cohort  as compared to patients who were homozygous for the major allele. By contrast, SNPs in the DDAH2, AGXT2, PRMT1, or NOS3 genes showed no significant associations with DCI.All three SNPs in the DDAH1 gene were significantly associated with the incidence of DCI. In each of the three SNPs, patients with DCI more frequently carried the minor allele than patients without DCI . Patientp = 0.011) and for poor neurological outcome at ICU discharge , p < 0.0001) and after 3 months , p = 0.001). No significant different was observed for the relative risk of death and vasospasm, respectively , ectively . Patientectively .Our study has three major findings: Firstly, the plasma concentrations of ADMA and SDMA and the CSF concentration of SDMA at the time of admission are positively associated with the incidence of DCI after subarachnoidal hemorrhage. Secondly, common genetic polymorphisms in the DDAH1 gene are responsible for differences in baseline ADMA plasma concentration, and common genetic polymorphisms in the AGXT2 gene are responsible for differences in plasma SDMA concentration; by contrast, the CSF concentrations of these metabolites were not significantly associated with these SNPs. Thirdly, and most importantly, patients with DCI more frequently carried the minor alleles of any of the three common DDAH1 SNPs that we analyzed; patients homozygous for the minor allele of DDAH1 rs233112 had a significantly higher probability of DCI than patients homozygous for the major allele. Patients carrying the DDAH1 haplotype with the most minor alleles had the highest relative risk for DCI and poor neurological outcome as compared to patients homozygous for the major allele of all three DDAH1 SNPs.ADMA is an endogenous inhibitor of nitric oxide synthesis. Its plasma levels are positively associated with the incidence of major adverse cardiovascular events and mortality . By inhiIn a previous genome-wide association study, we demonstrated that plasma ADMA concentration is associated with the DDAH1 gene, whilst SDMA plasma concentration is associated with the AGXT2 gene in the general population . In our Another observation of our study is that the plasma concentrations, but not the CSF levels, of ADMA and SDMA were significantly associated with the DDAH1 and AGXT2 genes, respectively. DDAH1 is highly expressed in liver and kidneys  and AGXTSDMA does not interfere with NO synthesis directly, but it indirectly modulates NO generation by limiting the intracellular availability of L-arginine via inhibition of the cationic amino acid transporter . SeveralBeyond its association with ADMA levels, there was a strong association of the DDAH1 gene with outcome after SAH: All three analyzed DDAH1 SNPs were associated with the incidence of DCI, and as compared to a combined genotype (haplotype) containing homozygosity for all three SNPs, haplotypes with an increasing number of minor alleles showed increasingly worse outcome. Thus, functionality of DDAH1 may be an important, in part genetically influenced determinant of the incidence of cerebral ischemia after SAH.Whilst presenting some exciting observations, our study has limitations. Firstly, the small sample size limits our ability to extrapolate our findings to various levels of severity of SAH. Specifically, the significant deviation of allele distribution in our critically ill cohort from the expected allele frequencies may suggest that genes regulating vascular NO production may predispose for SAH. Clearly, larger cohorts need to be genotyped to confirm and extend our findings. Secondly, due to the strict limitations linked to a clinical study in critically ill patients, we had no opportunity to investigate the mechanisms that may underlie the possible differential regulation of plasma and CSF dimethylarginine levels. This might be better addressed in animal models.In conclusion, our study provides evidence that the L-arginine–ADMA–NO metabolic pathway is involved in the pathophysiology of cerebrovascular ischemic complications after subarachnoidal hemorrhage. The DDAH1 gene, probably via determining the metabolism of ADMA, is prominently involved in mediating this risk. Carriers of genetic variants of the DDAH1 gene that lead to malfunction of the enzyme may carry a genetic risk of DCI after SAH.


TIL grain iAs concentrations for flood treatments across both years ranged from 26 to 127 μg kg–1. Additionally, seven quantitative trait loci (QTLs) were identified in the mapping population associated with grain iAs. A subset of eight TILs and their parents were grown to confirm field-identified grain iAs QTLs in a controlled greenhouse environment. Greenhouse results confirmed the genotypic grain iAs patterns observed in the field; however, iAs concentrations were higher under greenhouse conditions as compared to the field. In the greenhouse, the number of days under AWD was negatively correlated with grain iAs concentrations. Thus, longer drying periods to meet the same soil VWC resulted in lower grain iAs levels. Both the number and combinations of iAs-affecting QTLs significantly impacted grain iAs concentrations. Therefore, identifying more grain iAs-affecting QTLs could be important to inform future breeding efforts for low iAs rice varieties. Our study suggests that coupling AWD practices targeting a soil VWC of less than or equal to 30% coupled with the use of cultivars developed to possess multiple QTLs that negatively regulate grain iAs concentrations will be helpful in mitigating exposure of iAs from rice consumption.Arsenic (As) accumulation in rice grain is a significant public health concern. Inorganic As (iAs) is of particular concern because it has increased toxicity as compared to organic As. Irrigation management practices, such as alternate wetting and drying (AWD), as well as genotypic differences between cultivars, have been shown to influence As accumulation in rice grain. A 2 year field study using a Lemont × TeQing backcross introgression line (TIL) mapping population examined the impact of genotype and AWD severity on iAs grain concentrations. The “Safe”-AWD [35–40% soil volumetric water content (VWC)] treatment did not reduce grain iAs levels, whereas the more severe AWD30 (25–30% VWC) consistently reduced iAs concentrations across all genotypes. The TILs displayed a range of iAs concentrations by genotype, from less than 10 to up to 46 μg kg Arsenic (As) exposure from rice consumption is a serious and growing concern as rice is a staple dietary crop for half of the world’s population and is cultivated worldwide. Rice has been identified as a major As exposure route for humans and is often the principal source of As where drinking water As concentrations are low . ConsumpAs is naturally present in soil but can be exacerbated by the use of contaminated irrigation water or anthropogenic sources  . Paddy r–1 for polished rice and 350 μg kg–1 for husked (brown) rice products , a water saving irrigation management method, can reduce grain As accumulation; however, the extent of grain As reduction appears to be highly dependent on the severity and timing of the soil dry-down period . A meta-Multiple studies have established that rice cultivars vary in As accumulation rates; however, the genetics that influence accumulation or exclusion of As are not as well understood. Using mapping populations such as chromosome segment substitution lines (CSSLs) to study grain As accumulation can facilitate identification of As-affecting quantitative trait loci (QTLs). Previous studies have identified QTLs for total As accumulation in shoots and roots after applying an iAs treatment , as wellindica) as the donor as described by 1. The experiment was conducted at the Dale Bumpers National Rice Research Center in Stuttgart, AR, during the summer of 2013 and 2014. The soil at the site is described as a Dewitt silt loam  with a total C content of 0.67% and a total N content of 0.085%. The naturally occurring As content of this soil has previously been reported to be between 5.5 and 6.8 mg kg–1 (–1 phosphorous (P205) applied in 2013 and 2014, respectively, along with 100 kg ha–1 potassium (K2O) and 11 kg ha–1 zinc. Pre-emergent herbicide was applied, and additional pesticide treatments were applied as needed throughout the growing season according to local recommendations. The study consisted of 117 and 121 TeQing-into-Lemont backcross introgression lines (TILs) (genotypes) in 2013 and 2014, respectively, along with six repeated check plots of the two parents, evaluated under two irrigation regimes. Each irrigation treatment was conducted in separate adjacent fields. Within each irrigation regime, the TILs were arranged as a randomized complete-block design with four replications; however, only one field replication was used for the grain As analysis (see below).A set of 123 CSSLs, developed from backcrossing “Lemont”  as the recurrent parent and “Teqing” . The field was then flooded for 3 days, drained, and allowed to dry to the target VWC again; cycles were repeated a total of four times during the season , with each experimental unit consisting of 10 plants having approximately 30 cm between plants. All fields were irrigated after planting to ensure uniform germination and stand establishment. Approximately 2 weeks after emergence, 56 kg N hae season . A POGO e season . Soil VWTwo plants per experimental unit were monitored for days to heading and were harvested individually at maturity for collection of all agronomic data for each of the four field replications. The plants were dried to approximately 12% moisture, threshed individually, and agronomic traits were determined as an average of the two plants per experimental unit, across the four field replications. The paddy rice samples were stored at 4°C prior to further analysis.3 for 90 min at 95°C. At the end of extraction, tubes were removed from the hot block and allowed to reach room temperature before filtering the extracts through Whatman Grade 40 filter paper into new 50 mL polyethylene tubes. Filters were rinsed twice with 0.28 M HNO3, and the filtrate was diluted to 20 mL with 0.28 M HNO3.Rough rice from one of the harvested plants for each experimental unit in one field replication per irrigation treatment was shelled using the Testing Husker  to produce between 3 and 10 g of whole-grain brown rice in 2013 and 2014. Because of low yields, in some cases both of the plants per experimental unit were combined prior to shelling to reach a minimum of 3 g of brown rice per genotype. The grain samples were ground to a fine powder using a UDY Cyclone Sample Mill  with a 0.8 mm screen. Ground samples were stored at 4°C for later analysis. iAs was measured following the protocol described in –1, iAs of 92 ± 10 μg kg–1, DMA of 180 ± 12 μg kg–1 and monomethylarsonic acid of 11.6 ± 3.5 μg kg–1) and in-house standards were run for quality assurance. Additionally, control blank, spiked samples, and random technical replicates of samples were also analyzed. In this study, the mean measured iAs for the NIST 1568b standard was 99 ± 2 μg kg–1, which is within 1 standard deviation of the certified value of 92 ± 10 μg kg–1.iAs in filtered extracts was measured by inductively coupled plasma atomic emission spectroscopy using a hydride generation flow injection method . Technic3  were grown concurrently in two adjacent greenhouses at the USDA-ARS, Beltsville Area Research Center in Beltsville, MD. Sterilized seeds were germinated and then planted in 72-cell trays in potting soil. Approximately 2 weeks later, a single seedling was transplanted to a 19-l bucket made of high-density polyethylene filled with soil collected from the plow layer of a field used in the 2013/2014 field study at the Dale Bumpers National Rice Research Center in Stuttgart, AR. The buckets were filled with soil to a depth of 27 cm, leaving approximately 10 cm above the soil surface to allow for watering.The eight TILs and two parents were grown in four biological replicates per greenhouse and evaluated using two water treatments (10 genotypes × 4 replications × 2 water treatments × 2 greenhouses). Pots were arranged in two randomized complete blocks per bench, with four benches per greenhouse. All pots on a bench were subjected to the same water treatment and treatments alternated by bench , for a total of two AWD and two flood benches per greenhouse. Benches in the two greenhouses differed in distance from the cooling pad, and for this reason, the order of treatments alternated between the greenhouses .–1) was applied as N fertilizer in a three-way split during the 2 months following transplanting.Two water management treatments were used: flood (continuously flooded control) and AWD during which two cycles of wetting and drying were completed . The tar–1.Soil VWC was monitored continuously using TDR probes  for each individual pot in the AWD treatment. Probes were installed to measure soil VWC at a depth of 0–10 cm. Soil VWC was monitored in one flood control pot per greenhouse. For AWD treatments, pots were drained, and when the soil in a pot reached the target VWC (± 1%), it was reflooded. Thus, each pot was reflooded based on their individually measured VWC, and reflooding dates varied by pot, which is in contrast to field conditions where average field VWC was used to decide when to reflood the entire field. The flooded state was held for 8–10 days to ensure soil water saturation before draining and starting the second AWD cycle. The two AWD cycles were completed prior to heading stage after which pots were reflooded until harvest. Soil samples from the greenhouse were also analyzed for total As. The samples were analyzed using the Niton XL3t XRF Analyzer  and measured 7.1 ± 1.3 mg kgn = 8 across the two greenhouses) were processed for iAs analysis as described above. Additional agronomic traits including tiller and mature panicle number, shoot biomass without panicles, and total grain yield were determined for each plant at harvest. Temperature, humidity, and light measurements were also recorded for each greenhouse.Plants were harvested at maturity, and grain samples for each biological replicate  with a p < 0.05 and using Tukey HSD for multiple mean comparisons using a p < 0.05. Individual genotype comparisons between treatments (flood and AWD) were tested for significance using Welch t-tests.All data analyses were conducted in R (version 3.6.1)  and IciMIn 2013, the targeted average soil VWC was achieved at 40%, which is near saturation (termed Safe-AWD) for this soil type. There was little rainfall prior to heading, which occurred during the week prior to the fourth AWD cycle, but more rainfall occurred during the later grain fill stage . BecauseThe frequency distribution of brown rice iAs concentrations varied by water treatment and year . In 2013No QTLs were identified by irrigation treatment when data were pooled across years because of the large environmental variation between the 2013 and 2014 field seasons. Thus, data were grouped by each year and treatment combination for QTL identification. Seven QTLs affecting grain iAs accumulation were identified in the TIL mapping population corresponding to chromosomes 4, 5, 8, 9, 11, and 12 . Two QTLTo examine which of the identified QTLs are most abundant in the TILs, we calculated the percent of lines containing each QTL among four grain iAs groups defined by different ranges in iAs concentrations . Groups To examine whether the presence of other TeQing background introgressions, not specifically identified as iAs affecting QTLs, was influencing the observed grain iAs concentrations, the effect of TeQing kinship  was explored. However, there was no correlation between iAs accumulation and how closely related a TIL is to TeQing . Thus, tTIL Selection for Greenhouse Study) were examined separately, and a similar pattern was observed where grain iAs levels increased with increasing number of QTLs identified. The relationship was again more apparent under flood and the 2013 Safe-AWD treatments as compared to the more severe 2014 AWD30  reductions in grain iAs accumulation under AWD20 as compared to flood management practices .The eight selected TILs and their parents grown in the greenhouse  across almost all genotypes, with TIL634, a high iAs accumulator, in greenhouse 1, the only exception having a reduction of 36% in grain iAs concentrations under AWD. TIL634 had some of the lowest reductions in grain iAs from flood to AWD treatments overall. In contrast, the low iAs accumulator TIL455 had some of the greatest iAs reductions from flood to AWD with a mean reduction across both greenhouses in 2019 of 61%. In 2014 under AWD30, four TILs, 552, 596.11, 604.11, and 381.1, reduced grain iAs accumulation by at least 80%, whereas under the more severe AWD20 in 2019, the same TILs on average reduced iAs by markedly less, ranging from 49 to 64% on average across two greenhouses.Genotype differences in mean grain iAs levels within treatments were also examined, and some were found to be significant under flood (greenhouse 1) and AWD (greenhouse 2) (data not shown); however, the most significant differences were observed between the AWD and flood treatments. To compare iAs reductions between treatments, we calculated the percent change in grain iAs accumulation between flood and AWD in all studies . The fou–1 as compared to 31–110 μg kg–1 under slow AWD.In the greenhouse study, grain iAs accumulation was significantly negatively correlated to the total number of days a plant was under AWD to achieve the target soil VWC of 20%  . In genePot distance from the cooling pad  influencp = 0.029) in yield under AWD as compared to flood .In the greenhouse study, there was a trend for genotypes with increasing number of As accumulating QTLs to have higher mean total yield , mature A significant, strong correlation was found between grain iAs accumulation and total yield in the greenhouse AWD treatment  but was Mitigation of As accumulation in rice is dependent on the severity of AWD cycles. Several studies have shown that the more severe the AWD, the greater the reduction in both total As  and iAs The AWD implemented in the greenhouse pots was more severe (20% VWC) and controlled than that realized in the field (30–40% VWC). Additionally, the measurement depth for soil VWC in the greenhouse (10 cm) as compared to the field (5 cm) likely compounded the difference in severity of the AWD cycles experienced by the plants in the greenhouse vs. those in the field. In fact, AWD severity is the driving factor reducing grain As; however, the timing of AWD cycles is an important factor for yield and may also play a role in grain As reduction. A handful of studies have examined how the number and timing of AWD cycles impact total and iAs reduction. A study by p < 0.0001 for greenhouse 1 and p = 0.001 for greenhouse 2) between the number of days under AWD and grain iAs accumulation, even though all plants were subjected to the same AWD severity. This relationship was stronger in greenhouse 1 where the AWD was faster (R2 = 0.52) compared to greenhouse 2 (R2 = 0.23) where the AWD was slower, likely in part because the AWD tables in greenhouse 1 were farther from the cooling pad than in greenhouse 2  to the low iAs accumulator parent, Lemont (mean grain iAs levels of 34.7 μg kg–1 under AWD and 46.4 μg kg–1 under flood). Genotypic differences were also observed within the TIL population, with TIL455 and TIL634 displaying a mean difference of 40 μg kg–1 under flood and 50 μg kg–1 under AWD, respectively.A number of studies have shown that rice genotypic differences influence grain As accumulation and speciation . WilliamSafe-AWD . Concent in 2014 . Within  in 2014 . This is in 2014 . In our The 10 genotypes, two parents and eight TILs, grown across all experiments showed consistent patterns  across both field years and greenhouses under flood and AWD conditions . HoweverRoot biomass, physiology, and aeration capacity are also known to impact total and iAs accumulation in rice grain . Jia et Interestingly, we saw a strong, positive correlation between yield and grain iAs concentrations in the greenhouse study under AWD . Norton We identified seven QTLs in the TIL mapping population using the grain iAs data from the field 2013 and 2014 flood treatments . One of The TILs selected for the greenhouse study represent genotypes with low to high levels of grain iAs accumulation and contained differing numbers and combinations of QTLs. Although the identified QTLs did not overlap between the 2013 flood and 2014 flood datasets, we were still able to confirm that TILs harboring these QTLs, regardless of the treatment or year they were identified in, resulted in differences in grain iAs levels in the greenhouse study. These results support our finding from the field data that the identified QTLs are indeed affecting grain iAs.OsABCG22 (LOC_Os09g29660), an ATP-binding cassette (ABC) transporter gene, was identified in the region of QTL C9_18034390. This gene belongs to the ABCG-WBC cluster, which has been shown to be upregulated by submergence  and high concentrations of As (OsPLL4 gene (LOC_Os04g05050), a pectate lyase-like gene colocated with QTL C4_2481896, is highly expressed during panicle development (OsOFP18 (LOC_Os11g05770), colocated with QTL C11_2659978, is expressed both during seed development and during the young root stage (OsGLP8-1 (LOC_Os08g08920) was identified within the QTL region of C8_5186967; GLPs have been shown to colocate with other QTLs associated with drought and blast resistance (OsWRKY64 (LOC_Os12g02450) has been found to be upregulated under conditions of iron excess/toxicity and is thought to regulate root elongation as well as shoot growth under stress , resulting in consistently lower grain iAs concentrations when introgressed into Lemont, which has a moderately low iAs grain phenotype. Additionally, neither TIL had significantly reduced yield under AWD20. However, yields for TIL455 were higher under AWD (greenhouse mean of 35 g) compared to TIL 552 (greenhouse mean of 13 g), indicating TIL455 may hold promise for future studies focused on breeding low iAs varieties that are robust to severe AWD treatments.Grain iAs accumulation is dependent on both irrigation management practices and genotypic differences. While a severe AWD will reliably reduce grain iAs accumulation, incorporation of selected QTLs can ensure genetic mitigation of iAs, which is particularly important under field production conditions where soil moisture conditions may vary because of management systems or weather conditions. Ultimately, coupling AWD, or other water-saving irrigation management practices, with the deployment of low iAs accumulating cultivars is key to reducing rice iAs exposure while maintaining crop productivity.The original contributions presented in the study are included in the article/CF-B, JB, and AM conceived and designed the experiments. CF-B and JB performed the experiments. CF-B, JB, JE, and AM analyzed the data. JB, AM, EC, and VR provided the financial and technical assistance. CF-B wrote the initial manuscript. CF-B, AM, JE, EC, VR, and JB approved the manuscript for publication. 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.


In most guidelines, upper rectal cancers (URC) are not recommended to take neoadjuvant or adjuvant radiation. However, the definitions of URC vary greatly. Five definitions had been commonly used to define URC: 1) >10 cm from the anal verge by MRI; 2) >12 cm from the anal verge by MRI; 3) >10 cm from the anal verge by colonoscopy; 4) >12 cm from the anal verge by colonoscopy; 5) above the anterior peritoneal reflection (APR). We hypothesized that the fifth definition is optimal to identify patients with rectal cancer to avoid adjuvant radiation.The data of stage II/III rectal cancer patients who underwent radical surgery without preoperative chemoradiotherapy were retrospectively reviewed. The height of the APR was measured, and compared with the tumor height measured by digital rectal examination (DRE), MRI and colonoscopy. The five definitions were compared in terms of prediction of local recurrence, survival, and percentages of patients requiring radiation.A total of 576 patients were included, with the intraoperative location of 222 and 354 tumors being above and straddle/below the APR, respectively. The median distance of the APR from anal verge (height of APR) as measured by MRI was 8.7 (range: 4.5–14.3) cm. The height of APR positively correlated with body height . The accuracy of the MRI in determining the tumor location with respect to the APR was 92.1%. Rectal cancer above the APR had a significantly lower incidence of local recurrence than those straddle/below the APR (P=0.042). For those above the APR, there was no significant difference in local recurrence between the radiation and no-radiation group. Multivariate analyses showed that tumor location regarding APR was an independent risk factor for LRFS. Tumor height as measured by DRE, MRI and colonoscopy were not related with survival outcomes. Fewer rectal cancer patients required adjuvant radiation using the definition by the APR, compared with other four definitions based on a numerical tumor height measured by MRI and colonoscopy.The definition of URC as rectal tumor above the APR, might be the optimal definition to select patients with stage II/III rectal cancer to avoid postoperative adjuvant radiation. Preoperative chemoradiotherapy (CRT) has become an integral part of the multimodal treatment for stage II and III rectal cancer. Preoperative CRT has been shown to improve the local control and sphincter preservation rates, without significant effect on the overall survival (OS) and disease-free survival (DFS) –3. HowevAlthough the tumor height-related definitions provide a reproducible method for defining URC, the body habitus and sex must be considered during the assessment of tumor location as, for instance, the rectum is longer in taller patients . The decFive definitions of URC were used in this study and are as follows: 1) tumor >10 cm from the anal verge by MRI; 2) tumor >12 cm from the anal verge by MRI; 3) tumor >10 cm from the anal verge by flexible colonoscopy; 4) tumor >12 cm from the anal verge by flexible colonoscopy; 5) rectal tumor above the APR  adult patients (> 18 years) of either sex with histopathologically confirmed rectal adenocarcinoma; 2) tumor within 15cm from the anal verge by flexible colonoscopy; 3) pathological stage II (T3-4N0M0) or stage III (T1-4N1-2 M0) rectal cancer; 4) underwent curative resection of primary rectal cancer; 5) without preoperative CRT.Exclusion criteria: 1) patients who underwent palliative resection; 2) positive resection margin ; 3) synchronous or metachronous multiple primary colorectal cancer; 4) hereditary colorectal cancer syndrome; 5) previous history of pelvic radiation; 6) preoperative concomitant intestinal obstruction or perforation; 7) patients without recurrence who did not complete at least 24 months of follow-up after primary surgery.th edition). Resection margins, including circumferential, proximal, and distal margins, were considered positive if tumor cells were identified within 1 mm of the surgical resection margin.All surgeries were performed by seven chief surgeons, each with the experience of performing at least 100 operations for colorectal cancer per year, following a standardized operation protocol (including standard TME and high vascular ligation of the inferior mesenteric artery and vein). All resected specimens were examined using a standardized protocol that included TNM classification according to the American Joint Committee on Cancer-International Union Against Cancer ] was administered orally throughout the period of radiation treatment. Postoperative adjuvant chemotherapy (CapeOX or mFOLFOX6) was administered 3 weeks after the completion of radiation and continued for 4–6 months.For middle and lower rectal cancer, preoperative CRT was recommended to all patients with stage II/III tumor, and some patients refused. For upper rectal cancer, preoperative CRT was not recommended to patients with stage II/III tumor (excluding T4b). All patients with stage II/III rectal cancer who did not receive preoperative CRT were recommended to undergo postoperative CRT, and some patients refused to take postoperative CRT or chemotherapy. Postoperative adjuvant CRT was initiated 4 weeks after surgery and continued for 6 months. The dose of postoperative adjuvant radiation was 1.8 to 2.0 Gy daily for a total of 23 to 28 fractions over 5–6 weeks and resulted in a total dose of 46.0 to 50.4 Gy. Postoperative adjuvant radiation was delivered by the three-field or four-field box technique to the original area of tumor and mesorectum, presacral region, and the internal iliac lymph nodes. Postoperative adjuvant concurrent chemotherapy . Local recurrence and distant metastasis occurred in 1.4% and 12.0% of patients at 2 years, respectively. The actual 2-year rate of OS, DFS and LRFS were 95.0%, 86.8% and 91.5%, respectively.P=0.042, Rectal cancer above the APR exhibited a significantly lower incidence of local recurrence than those straddle/below the APR . The tumor height measured by DRE was correlated with that measured by colonoscopy in 394 cases ]  compared with the other four definitions using a numerical tumor height measured by MRI and colonoscopy  for females, and 9.7 cm (7–16 cm) for males . In a KoThe APR divided rectal cancer into two subtypes: intraperitoneal and extraperitoneal. The local recurrence rate has been found to be much lower in intraperitoneal compared to the extraperitoneal rectal cancer patients  and is cRigid sigmoidoscopy is recommended for measuring the height of rectal cancer, but it is performed in only a minority of patients  and is nOur results demonstrated that the APR is a distinct and individualized landmark that can be easily identified by preoperative MRI and intraoperative finding. Patients with rectal cancer above the APR exhibited a lower incidence of local recurrence, and there was no significant difference between the radiation group and the no-radiation group in terms of OS, DFS and LRFS. Tumor location with respect to the APR is an independent predictor of LRFS, while other subdivisions based on a fixed distance measured by DRE, MRI or colonoscopy were not associated with survival outcomes. Therefore, the definition of URC as a rectal tumor above the APR is superior to other definitions, based on a fixed tumor height as measured by MRI and colonoscopy, for selecting patients with stage II/III rectal cancer that should avoid radiation. This definition will not only help us to select suitable cases that should undergo radiation, but also to reduce the incidence of radiation-related toxicity and medical expenses.There are several limitations in our study. First, this was a retrospective study. In clinical practice, physicians may have preferred to recommend postoperative radiation to high risk patients. Therefore, selection bias is unavoidable. Second, patients with positive resection margin were excluded in this study. Adjuvant radiation might also be needed for some URC patients receiving R1/R2 resection. Third, the included patients did not receive preoperative CRT as recommended by the NCCN and ESMO guidelines due to various reasons. In Asian countries , postoperative adjuvant CRT or adjuvant chemotherapy is considered the treatment of choice for stage II or III rectal cancer, especially for low risk cases and those with URC . Fourth,The definition of URC as a rectal tumor above the APR might be better than other definitions based on a numerical tumor height measured by MRI and colonoscopy in selecting patients with stage II/III rectal cancer to avoid adjuvant radiation. The tumor location relative to the APR could be recorded in the preoperative rectal MRI imaging, intraoperative surgical records and postoperative histopathology reports for prognostication and treatment planning . However, further prospective RCTs with a larger sample size are required to validate the findings of this study.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethical committee at Changhai Hospital.FS, LL, and WZ conceptualized the study. XG, BZ, and JL conducted the data curation and wrote the original draft. XG and JL performed the formal analysis. XG acquired the funding. JK, HG, CB, ML, and SZ conducted the investigation. XG developed the methodology. FS and WZ provided the resources. FS, LL, and WZ supervised the study. XG, BZ, JL, JK, HG, CB, ML, SZ, FS, LL, and WZ wrote, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by Shanghai Pujiang Program (#2019PJD052) and the National Natural Science Foundation of China (#81572332).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.


LEP, STAT5A, PRL, and GHRHR were significantly associated with phenotypes. This study provides first insights into the genetics of milk and growth traits in the Barki sheep. The findings concluded that LEP, STAT5A, PRL, and GHRHR might be regarded as candidate genes to improve the Egyptian Barki sheep breed. The Barki sheep breed is one of the main sheep breeds in Egypt, and it is well adapted to the harsh desert conditions in the Mediterranean zone. Growth performance has an important role regarding the supply of red meat for human consumption. In addition, milk production is very important to adequately feed newborn lambs and prevent them from starvation. In this study, segregating single nucleotide polymorphisms (SNP) were identified in the coding regions of eight candidate genes for growth and milk traits by direct sequencing. Subsequently, a population of Barki ewes and lambs was screened for these SNPs, and associations between genotypes and traits of interest were assessed. Out of the candidate genes, SNPs of LEP, IGF1, DGAT1, STAT5A, PRL, CSN1S2, GHR, and GHRHR, of which one representative single nucleotide polymorphism (SNP) located in the coding region was selected for genotyping. Data from 251 Barki sheep were used in this study. Association analysis between SNPs and lamb growth traits identified rs420693815 of the LEP gene to be significantly associated with weaning weight and average daily gain. In ewes, significant effects on milk yield and composition have been estimated for LEP (rs420693815), STAT5A (rs161082816), PRL (rs422713690), and GHRHR (rs414991449). The results indicated that these genes might be considered as interesting candidates for further investigations to improve growth and milk performance in Barki sheep.The most common sheep breeds of Egypt are Ossimi, Rahmani, and Barki breeds. The latter one is well adapted to the challenging desert environment, characterized by food shortage and a high temperature fluctuation. Growth performance of Barki sheep has an important economic value in terms of minimizing the shortage of mutton meat in Egypt. Further, milk production is of great importance for feeding newborn lambs. Eight candidate genes, recently associated with production traits in different breeds, were used to study the effect of genotype on lamb growth and ewe milk traits. The examined genes were  PRL), leptin (LEP), insulin-like growth factor hormone 1 (IGF1), diacylglycerol O-acyltransferase 1 (DGAT1), signal transducer and activator of transcription 5 (STAT5), alpha (α)s2-casein (CSN1S2), growth hormone receptor (GHR), and growth hormone-releasing hormone receptor (GHRHR) [PRL can be used as a candidate marker associated with milk yield and milk composition traits [IGF family, IGF1 is considered an important factor associated with cell differentiation, embryogenesis, metabolism [DGAT plays an important role in triacylglycerol biosynthesis as well as milk and growth traits [STAT family has seven members , and STAT5 is known to play a central role in signal transduction from prolactin to milk protein genes [CSN1S2 alleles are associated with a normal (α) s2-casein synthesis level [GHR and GHRHR are valuable candidate genes. Polymorphisms in GHR are associated with traits related to growth performance, body size, and meat quality in cattle [FABP4 and calpastatin on some carcass traits of Barki lambs [Small ruminants play an important role in supplying meat and meat products in arid regions. Regarding the Egyptian agriculture sector, sheep production serves as a valuable source of income to farmers and as an important source of meat and milk . In Egyp (GHRHR) ,8,9. Pro (GHRHR) . Polymorn traits . Leptin n traits . It has n traits  and in Mn traits . As a metabolism ,15, reprtabolism ,17. Therh traits . The STAin genes . Caseinsin genes . The casin genes ,22. CSN1is level . The gron cattle ,25,26,27n cattle . Concernki lambs . Heritabki lambs . TherefoSamples and data were collected from the farm of the Matrouh Resources Project (location 1) and the Maryout research station (location 2) that belongs to the Desert Research Centre (DRC), Ministry of Agriculture, Egypt. The experiment was carried out according to all ethics and animal rights (DRC) considering all regulations in conformity with the European Union Directive for the protection of experimental animals (2010/63/EU).Phenotypic and genotypic data from 111 Barki ewes and 140 of their lambs (44 ewes and 66 lambs from location 1 and 67 ewes and 74 lambs from location 2) from one breeding season were used in this study. Ewes were randomly mated with certain rams of the same breed according to the normal farm practice. No records were available for the rams. Animals were kept under an intensive production system and were housed in semi-open yards throughout the experimental period. The lambs were kept all day with their dams for suckling until weaning at three months of age, respecting the natural ewe−lamb relationship. Some of the examined lambs were not the offspring of the ewes under study. Furthermore, some lambs have no data for their mother’s milk. Ewes in the two locations were fed daily on a feed concentrate (0.75 kg per head) and clover hay (0.5 kg per head) during the experimental period, and lambs were fed daily only on their dam’s milk from birth to weaning age at three months of age. Fresh water was available to sheep ad libitum.Live body weight for each lamb was recorded at birth and weaning by electronic balance. Average daily gain was calculated for every lamb. Milk yield was recorded biweekly from the time of parturition until weaning using hand milking technique. Lambs were separated from their dams 12 h before milking. The ewe was milked in the morning; another milking was carried out in the evening by the same approach. Milk yield per day was measured in mL by summation of the morning and evening milking. Total milk yield was calculated by summation of the daily milk yields for 90 days. Milk samples were stored at −20 °C and chemically analyzed to determine the percentages of fat, protein, lactose, and total solids using milko-scan .Blood samples were collected from the jugular vein using test tubes containing disodium ethylene diamine tetra acetic acid (EDTA-Na2). All blood samples were stored at −80 °C until DNA extraction. DNA was extracted by using a commercially available kit according to the manufacturer’s instructions . http://bioinfo.ut.ee/primer3-0.4.0/) according to the latest sheep genome information . The primer pairs used to detect SNPs are shown in Pooled DNA samples were prepared from five lamb samples and five ewe samples from different locations. The pooled samples were subjected to a polymerase chain reaction (PCR) to amplify specific regions of the candidate genes and to identify segregating single nucleotide polymorphisms (SNPs). The PCR assay was performed using respective primer sets in a total volume of 20 µL according to the manufacturer’s instructions . Gene-specific primers were designed with Primer3 software (v.0.4.0) , Lj is the effect of location (j = 2 levels), and eijk is the error effect. Another model was used to detect the effect of genotype on the lamb growth traits as follows: Yijkl= μ + Gi+ Lj+ SK+ eijkl, where Yijkl is the analyzed trait, μ is the overall mean, Gi is the effect of genotype (i = 3 levels), Lj is the effect of location (j = 2 levels), Sk is the effect of lamb sex (k = 2 levels), and eijkl is the error effect. p < 0.05 was considered significant. p < 0.1 was considered a tendency for significance. The Hardy–Weinberg equilibrium (HWE), polymorphic information content (PIC), heterozygosity (He), and homozygosity (Ho) were tested for all alleles by using the Cervus (V3.0.7.0) program . The assDescriptive statistics of total milk yield in three months and percentages of milk components including fat, protein, lactose, and total solids are shown in LEP, rs400398060 in exon 3 of IGF1, rs409119650 in exon 9 of DGAT1, rs161082816 in exon 11 of STAT5A, rs422713690 in exon 3 of PRL, rs420391387 in exon 8 of CSN1S2, rs413776054 in exon 10 of GHR, and rs414991449 in exon 13 of GHRHR. Results of Hardy–Weinberg equilibrium for all selected SNPs are shown in p > 0.05). rs409119650 and rs161082816 were in low polymorphic information content status (PIC < 0.25), while rs420693815, rs400398060, rs422713690, rs420391387, rs413776054, and rs414991449 were in moderate polymorphic information content status (0.25 < PIC < 0.50). The homozygosity of all loci sites was higher than the heterozygosity, except for rs420391387 and rs422713690, where the homozygosity was equal to the heterozygosity. In lambs, the selected SNPs for all candidate genes were in Hardy–Weinberg equilibrium status . Ewes with TT genotype had a higher milk yield than ewes with GT and GG genotypes. Ewes carrying the GT genotype had a higher fat percentage than ewes with GG and TT genotypes. For rs161082816 of STAT5A, the milk of ewes with AA genotype were significantly (p ≤ 0.05) higher in lactose percentage compared to ewes with GG and AG genotypes. For rs422713690 of PRL, a significant association with milk yield (p ≤ 0.1) was observed. Ewes with GG genotypes had a higher milk yield than ewes with AG and AA genotypes. rs414991449 of GHRHR was significantly associated with total solids percentage (p ≤ 0.05) and with protein percentage (p ≤ 0.1). Ewes having TT genotypes had higher total solids and protein percentages than ewes with CT and CC genotypes.LEP as significantly associated with weaning weight and average daily gain (p ≤ 0.1). Lambs with GT and GG genotypes had a higher weaning weight and average daily gain than lambs with TT genotype. The other selected SNPs in the candidate genes showed a non-significant association with growth traits (p ≥ 0.1).The results of the association analysis of SNPs with lamb growth traits are summarized in LEP were significantly associated with weaning weight and average daily gain. Interestingly, rs420693815 had also a significant effect on milk traits comprising milk yield and fat percentage in the Barki ewes. The results indicated the inverse relationship between milk yield and fat percentage. Ewes with the highest milk yield had the lowest fat percentage [LEP is considered as one of the candidate genes affecting body fat content [LEP might be of relevance in mobilizing lipids for, e.g., milk production with possible implication on the offspring’s body weight. In agreement, genetic variants of leptin have been shown to influence milk performance in cattle [LEP polymorphism was found to be significantly associated with milk yield in Najdi ewes of Saudi Arabia [LEP in growth traits [LEP (rs420693815) is causative for the effects or acts as tagging SNP in linkage disequilibrium with the causal one. However, LEP as candidate gene might be further considered as a locus for improving performance and production traits in the breeding programs of Barki sheep. Furthermore, analyzed SNPs in STAT5A, PRL, and GHRHR revealed a significant association with milk production traits in the Barki ewes. For PRL (rs422713690), animals with the heterozygous AG genotype showed lower milk yields than homozygous animals. PRL is a hormone released from the anterior pituitary gland and acts to initiate and maintain lactation [PRL gene is located on the ovine chromosome 20 where putative quantitative trait loci for milk yield, fat, and protein percentage are located [PRL has been shown to affect all these traits in Serra da Estrella sheep [PRL might act as a marker gene for milk production traits also in the Barki sheep. Regarding the investigated SNP in STAT5A (rs161082816), Barki ewes which carried A alleles showed higher milk lactose percentages compared to animals exhibiting G alleles. STAT5A is a key player in mammary gland development [STAT5A is known to mediate PRL and GH signals via transcriptional stimulation of gene expression in milk-secreting mammary epithelial cells. Due to its prominent role in milk production traits, STAT5A has been previously investigated in cattle and goat and genetic variants have been associated with milk fatty acid profiles and milk yield [STAT5A as a promising candidate gene for further analyses of milk traits in Barki sheep. The SNP located in GHRHR (rs414991449) was significantly associated with the percentages of total milk solids and milk protein, whereby the appearance of the T allele prompted the highest values. In fact, GHRHR mediates effects of its ligand growth hormone-releasing hormone (GHRH) to regulate growth hormone (GH) synthesis and secretion [GHRHR might therefore impact on GH axis signaling as it has been shown for body growth in humans and mice [GH locus has been associated with milk traits such as milk fat percentage and milk yield [GHRHR on GH signaling affecting milk production and composition. Results did not support any significant associations of sequence variants of IGF1, DGAT1, CSN1S2, and GHR with growth or milk traits in the studied Barki population. This might also be related to the fact that for some of the SNPs, a low representation of alternative homozygotes was found in the studied population. However, associations of segregating SNPs in IGF1 and DGAT1 with growth, milk, and wool performance traits have been described in various sheep breeds such as Makeoi, Baluchi, Hu, Sarda, and Mehraban [In this study, a representative SNP of each of the selected functional candidate genes was associated with growth and milk production traits obtained from Barki lambs and ewes. Genotyping results showed that none of the selected SNPs deviates from HWE. These results indicated for the absence of strong selection pressures, probably due to the coherent housing environment and the lack of artificial selection. These facts might contribute to a stability of allelic and genotypic frequency for a long time. Results of the polymorphic information content state and homozygosity-to-heterozygosity relationships confirmed that an inbreeding scheme was applied at the different locations creating a high genetic variation between populations and lower genetic variation between individuals in the same population. These results suggest that an application of selection employing genomic information will be effective in the respective population. However, the relatively low sample size, due to the lack of management with breeding programs and routine sampling in the Barki sheep, represents a certain limitation for the genetic evaluation in this study. The results of association of SNPs with lamb growth traits showed that rs420693815 of rcentage . Accordi content . Through content ,35. Due n cattle . A LEP pi Arabia . Moreoveh traits . With reactation . The PRL located ,42. Indela sheep  and milkelopment . In partlk yield ,46,47. Tecretion ,49,50. Gand mice ,52. In slk yield ,54. CorrMehraban ,60,61,62LEP, STAT5A, PRL, and GHRHR were significantly associated with lamb growth and ewe milk traits, while IGF1, DGAT1, CSN1S2, and GHR genes showed no significant associations. LEP, STAT5A, PRL, and GHRHR might be considered as interesting candidate genes for further investigations to improve growth and milk performance in the Barki sheep.A SNP-trait association analysis was performed to study the effect of genotype on growth and milk performance traits in Egyptian Barki sheep. Results concluded that the selected polymorphisms in


Probe of pH study may kink in the esophagus leading to nasal symptoms during the removal. Probe of pH study may kink in the esophagus leading to nasal symptoms during the removal. Probe movements may complicate the testing leading to digestive symptoms and nose pains during the removal of the probe.Hypopharyngeal multichannel intraluminal impedance‐pH monitoring (HEMII‐pH) with nasopharyngeal catheter placement is the standard of care for the diagnosis of gastroesophageal reflux disease (GERD) or laryngopharyngeal reflux (LPR).The aim of this paper was to report two unusual complications of HEMII‐pH.2A 65‐year‐old patient was referred to the Reflux Consultation of the Elsan Polyclinic (Poitiers) for a chronic history of belching, postnasal drip, dysphonia, globus, and heartburn. The nasofiberoptic examination found posterior commissure hypertrophy and arytenoid erythema. The history of the patient included refractory gastroesophageal reflux disease (GERD), while the gastrointestinal (GI) endoscopy reported Hill 2 hiatal hernia and esophagitis (grade A). Regarding the laryngopharyngeal complaints and the previous resistance to proton pump inhibitors (PPIs), a HEMII‐pH testing  was realized to confirm the diagnosis. The HEMII‐pH was composed of 8 impedance segments and 2 pH electrodes. The catheter model used was introduced transnasally and considered the esophageal length of patient (GI endoscopy/manometry). Six impedance segments were placed along the esophagus zones Z1 to Z6), and they were centered at 19, 17, 11, 9, 7, and 5cm above the lower esophagus sphincter (LES). Two additional impedance segments were placed 1 and 2 cm above the upper esophagus sphincter (UES) in the hypopharyngeal cavity. The two pH electrodes were placed 2 cm above LES and 1‐2 cm below UES. The probe was fixed to an external electronic data recorder that monitors the esophageal pH. The association between symptoms and reflux episodes was studied: Patient recorded the time of meals and the occurrence of key symptoms  through the HEMII‐pH device. The patient came back 24 hours after the placement of HEMII‐pH catheter to remove it. He reported the occurrence of dysphagia 2 hours after the probe placement. The removal of the probe was associated with nose pain, and the otolaryngologist discovered a distal probe node  and the characteristics of the device were similar to those reported in the first case. The patient had belching, nausea, and several vomiting episodes during the probe placement. During the 24‐hour testing, the patient reported many heartburn and belching episodes, which were not reported in the initial clinical picture. The removal of the probe was associated with nose pain, and the physician found a folded probe (above Z5 segment; Figure 4The most common complications/adverse effects of HEMII‐pH consist of nose pain, runny nose, nose bleeding, throat pain/discomfort, chest pain, cough, headache, and dysphagia.5In this paper, we reported two very rare complications of HEMII‐pH, consisting of probe movements and node in the esophagus. These complications may affect the tracing analysis if the HEMII‐pH testing is realized for GERD. For LPR, the impact is lower because the LPR diagnosis is mainly based on the occurrence of hypopharyngeal reflux episodes.None declared.JRL: wrote the paper, analyzed the case, conducted literature review, and performed the research. FB and SS: analyzed the case and wrote the part: “case report.”Patient consented to the publication.