Pituophis ruthveni), which is a threatened North American colubrid. Using ultrasound, we described 7 different stages of the follicles and developed markers of progressive follicular maturation for this species, moreover, we dissected two specimens to describe the macroscopic anatomy of the female reproductive system. Furthermore, we identified the correspondence of the sexual activity peak with a particular follicular stage. This data provides parameters for evaluating growth patterns of ovarian follicles in relation to maturation, ovulation, fertility, and reabsorption that can be useful for conservation of this species as well future conservation programs, commercial breeders, and clinicians monitoring reproductive health and breeding of snakes.A detailed knowledge of reproductive biology is of paramount importance for establishing ex situ conservation programs. This paper describes in detail the follicular morphology, ovarian cycle and reproductive anatomy of the female Louisiana pinesnake (Pituophis ruthveni). Ultrasound procedures were performed weekly for 11 females during the 2020 reproductive season by submerging the last half of an unanesthetized female in water and using a 3.0–10.0 MHz linear array transducer placed and moved along the gastrosteges to explore the whole reproductive tract. The presence of follicles, their size, echogenicity, and stage of development was assessed. We observed small, round, anechoic, linearly aligned previtellogenic follicles in the coelom at the beginning of the reproductive season and found that structures dramatically increased in size and shifted in echogenicity as follicles matured and developed before and after ovulation. We classified follicles according to ultrasonographic appearance into 7 different follicle categories: previtellogenic, early vitellogenic, vitellogenic, preovulatory, peri-ovulatory, post ovulatory, and shelled. Using ultrasound, we developed markers of progressive follicular maturation for the Louisiana pinesnake and identified signs of abnormal development and post ovulatory follicle reabsorption. Detailed description of follicular maturation will be useful to improve captive breeding successes, identify mechanisms of reproductive failure, and develop artificial insemination.Accurate monitoring of reproductive activity is necessary for success of captive breeding and recovery of endangered species. Using ultrasonography, we aimed to describe the stages of follicle development of the endangered Louisiana pinesnake ( Biodiversity is declining at an alarming rate across the globe. Captive breeding programs are one tool in a multilateral approach to the conservation and recovery of imperiled species ,2. WhilePituophis ruthveni) is a large, cryptic colubrid listed as threatened under the United States Endangered Species Act (USFWS 2018) and as Endangered by the International Union on the Conservation of Nature (IUCN) Redlist [Pinus palustris) [The Louisiana pinesnake (USFWS 201 and as Elustris) ,10,11. HThe characterization of female reproductive cycles and the identification of biomarkers of female receptivity are necessary to set the timing of pairings for natural breeding and the development of artificial insemination protocols. Although there is a general understanding of the seasonal reproductive cycle of the Louisiana pinesnake, a detailed description of each stage in the reproductive cycle of females is lacking for this species. Based on population movement data, natural breeding of Louisiana pinesnakes in the wild is thought to occur between mid-April and early June . In captAbdominal palpation is the most widely used technique to assess broad- scale reproductive state of snakes ,14 and hPituophis ruthveni) housed in a Louisiana Pinesnake Breeding facility in Memphis Tennessee, USA. Four individuals were first time breeders and seven were multiparous. Weight of the animals ranged from 1.17 kg to 2.45 kg (mean 1.65 ± 0.48). All procedures and experimentation were performed in accordance with the guidelines of the IACUC committee of the Memphis Zoo (study #2018-1 and 2020-4). At the start of the study, all females were determined to be healthy with no signs of disease or illness upon clinical examination. The females were housed separately in individual Neodesha plastic cages, with newspaper substrate, and provided large hides and free access to water. Females were fed weekly unless otherwise specified. To simulate the natural seasonal cycles of the temperate latitude of in situ populations, we provided a cooling period to simulate brumation for approximately 90 days. This cooling period was performed according to Louisiana pinesnake SSP captive breeding recommendations. Beginning on 1 December 2019, building temperatures were lowered from 28.3 °C to a minimum of 10 °C. This was accomplished by slowly reducing temperatures by up to 10° every 3–5 days until the minimum temperature of 10 °C was reached. Once the minimum temperature was reached, building temperatures were allowed to fluctuate between 8.9 to 15 °C based on external temperatures, with heating and cooling provided as needed to maintain a mean temperature of 10 °C. Temperature reduction was finalized by 15 December, at which point the artificial lights were turned off. Windows along the outside walls of the building were opened to both provide the appropriate photoperiod cycle and improve airflow through the building to encourage natural shifts in temperature throughout the brumation period. On 2 March 2020, the windows were closed, the lights were turned on (12 h:12 h), and the minimum temperature was increased from 10 °C to 18.5 °C. The temperature was increased by up to 10° every 3–5 days until a minimum temperature of 28.3 °C was reached. By 16 March, the temperatures were allowed to fluctuate between 26.7–29.5 °C with a mean temperature of 28 °C, and the photoperiod was set to a 13 h:11 h cycle. Feeding was resumed once temperatures were at least 25.5 °C and the snakes were visibly active. Each adult snake was offered 2–3 small to medium-sized rats on a weekly basis, with feeding amount adjusted based on the prey response of each individual snake. Throughout the breeding season the building was then allowed to continue to heat naturally to 28.3–29.4 °C, and then air conditioned as needed to maintain 28.3 °C.Throughout the breeding season in 2020, we tracked the ovarian cycle of 12 mature female Louisiana pinesnake  was used for the ultrasound examination. The snakes were manually restrained and the caudal third of the animal was sunken in shallow warm water (28–33 °C), to remove any air trapped between the scales impeding correct visualization ,17. A 5.Abdominal palpation was routinely performed starting the first week of April by a single experienced operator (S.B.R.). Snakes were fasted at least 3 days prior to palpation to prevent regurgitation and avoid food items in the digestive tract while palpating for follicles. Abdominal palpation is a widely used technique to assess reproductive state of snakes ,14 and hTo verify anatomical information obtained via ultrasound imagery, the reproductive anatomy of two mature female Louisiana pinesnakes, proceeding from a different institution and deceased for natural reasons, were dissected. The coelomic cavity was accessed cutting the skin between the second and third row of scales on the right lateral body wall and dissecting the gastrosteges and abdominal muscles. Ovaries were readily identified and the mesovarium isolated and dissected. Ovaries, oviduct, and cloaca were then isolated. A 3 French urinary catheter was then inserted into the vaginal pouch to identify the path of the oviducts . Finallyhttps://www.R-project.org accessed on 30 September 2022) and alpha was set at 0.05. Summary statistics are presented as means ± standard deviation.Sonographic appearance and size (cm) of structures, palpation findings, as well as dates of mating, skin shedding, egg lay date, and finally numbers of fertile and infertile eggs laid were recorded. The size of follicles at each category of follicular development was compared using a Kruskal–Wallis non-parametric statistical test followed by pairwise comparisons using a Dunn’s post hoc test with a Benjamini-Hochberg correction for multiple comparisons . SeparatSnakes did not show any sign of distress during the ultrasound examination. Manual restraint proved to be adequate for the procedure, and the water bath provided excellent image quality. The right ovary was echolocated immediately caudal to the gall bladder, while the left ovary was found to caudal and to the right. The follicular structures were then classified according to soft tissue echogenicity into 7 different categories: previtellogenic follicles, early vitellogenic follicles, vitellogenic follicles, preovulatory follicles, Peri-ovulatory follicles, post ovulatory follicles, and shelled eggs .Ultrasound images are presented in From 30 March to 12 May, early vitellogenic follicles B were viFollicles continued to elongate, and the anechoic pocket disappeared. We defined these hyperechoic structures with little to no anechoic pocket, and a thick hypoechoic outline occurring between the preovulatory stage and prior to the females having overt lower body swelling, as peri-ovulatory E, these n = 9) laid eggs 7.6 ± 2.2 days after shedding, between 31 May and 2 July. Immediately following oviposition, string-aligned, anechoic previtelogenic follicles were again apparent in all females assessed within 1 week of lay.From 4 May to 15 June post ovulatory follicles were present in conjunction with the female exhibiting a swollen lower body. Post ovulatory follicles are larger and started to develop hypoechoic and hyperechoic layers and the hypoechoic rim became thin. Within a few weeks after the follicles entered the oviduct, shelling became visible. Depending on the extent of calcification the shell was visible as a hyperechoic layer deposed around the hyperechoic egg A,B. Firsn = 9) oviposited 95 days (range 77–107 days) after the cooling period ended on 16 March and females developed preovulatory follicles 48.9 ± 7.9 days and peri-follicles 40.4 ± 8.1 days before laying eggs.The date of reproductive milestones (end of the cooling period (16 March), preovulatory, peri-ovulatory stage, and lay date for each female was recorded) and length of time between milestones are calculated and summarized in There were no substantial differences in follicular development found between first time breeders and previously bred females. Nine of 12 of the females laid eggs. Together the females produced 40 fertile of 63 eggs laid (63.5% fertile). We also found several instances of abnormal follicles in females. One female exhibited abnormal follicular distribution with follicles clustered instead of string-aligned in coelom during previtellogenic stage SB 164, . These fp < 0.001; Transverse length: H = 309.21, df = 6, p < 0.001; Longitudinal width: H = 197.64, df = 6, p < 0.001; Longitudinal length: H = 208.65, df = 6, p < 0.001). Post hoc comparison tests between developmental stages found most stages differed significantly based on size. However, comparisons of longitudinal measurements showed late stages of follicle development to be similar in size . No animal showed any signs of distress during the procedure and no snakes were injured. According to ultrasound observations, the stage and size of follicles at first detection by palpation varied by individual and ranged from vitellogenic (1.6–3.4 cm long) to post ovulatory stage (3.4–5 cm long), although presence of follicles was most frequently palpated after the pre-ovulatory stage. Follicle presence was determined for 40% of the females by the preovulatory stage, while detection of follicular presence increased to 80% relative to ultrasonography by periovulatory stage. The capability of determining the stage of the follicles was limited to determining relative size and hardness. An accurate count of follicles was possible during peri-ovulatory stage when follicles were 4–6 cm long and could still be felt as individual, distinct but soft follicles. As follicles continued to develop in the oviduct, the follicles become soft, enlarged to take up the most of the coelomic cavity. Palpation of distinct margins between follicles through the scales was lost at this stage and palpation was ceased. During the post ovulatory stage females became pear shaped and were considered gravid. Regular palpation was ceased as not to damage developing eggs or the distended oviduct.The female genital system of Louisiana pinesnake repeats the structure already described for other snakes ,23,25,26Pituophis ruthveni) which produces significantly larger eggs than other North American colubrids (Reichling 1990) and previously described oviparous snakes [n = 4). Females laid 30–40 days after observed mating (n = 9).Although the use of ultrasound has become more common to monitor reproductive status by commercial and private snake breeders there are still limited scientific references using ultrasound to detail follicular cycle of oviparous snakes ,20,21 ans snakes ,20,21 Fos snakes ,14,16,21s snakes . We obsen = 9). The mean length combined fertile and non-fertile eggs was 8.66 ± 0.99 cm (n = 51) at lay, indicating that ultrasound measurements at the end of gestation were predictive of egg size at lay. The mean live hatchling size at emergence from fertile eggs was 47.28 ± 3.8 cm . The size of laid eggs, hatchlings, and clutch were within ranges previously reported for the Louisiana pinesnake [Post ovulatory follicles during gestation elongated to a mean of 7.46 ± 1.94 cm in length and shelled in conjunction with females exhibiting signs of impending ecdysis. Shelled post ovulatory eggs reached 8.76 ± 1.68 cm in length on longitudinal plane with no fetal development visible. This length exceeds the maximum size reported for any snake species where follicle size has been followed through late gestation ,16,20,21inesnake . Louisiainesnake ,30 and pinesnake  could coUltrasound examination of ovaries in snakes has been described in the literature ,22, and Detailing the description of the echogenicity and sizing of follicular maturation of the Louisiana pinesnake using ultrasonography is vital knowledge to improve captive breeding efforts, identify mechanisms of reproductive failure, and continue to develop assisted reproductive technologies for this species. The effectiveness of our classifications to predict optimal timing for artificial insemination and natural mating are beyond the objective of this paper; however, we have developed a framework to assess this in future research. It is unknown if like mammals, optimal timing of fertilization for reptiles requires accurate timing of insemination relative to follicular maturation; however, improved outcomes of fertilization and hatch rate have been shown for corn snakes when follicle maturation was used to time artificial insemination . This stWe described the reproductive cycle of Louisiana pinesnake including follicular maturation, ovulation, and abnormal follicular growth and atresia as well as post-ovulatory egg development through pre-lay shed using ultrasonography. We detailed the related echogenic and size changes of 7 follicular stages throughout the entire reproductive cycle and this data provides parameters for evaluating growth patterns of ovarian follicles in relation to maturation, ovulation, fertility, and reabsorption that can be useful for captive breeders and clinicians monitoring reproductive health and breeding. Furthermore, we provided the first description of follicle size and appearance with this significant increase in follicular size correlated to overt breeding behavior and thereby presenting a potential optimal timing for artificially inseminating females during the reproductive cycle.


Weight management is strongly promoted for overweight or obese patients with type 2 diabetes (T2DM) by current guidelines. However, the prognostic impact of weight loss achieved without behavioural intervention on the mortality and cardiovascular (CV) outcomes in diabetic patients is still contested.We searched the PubMed, Embase, and Cochrane Library databases for studies that investigated the association of weight loss or weight variability with mortality and CV outcomes. Results of studies that measured weight loss by percentage weight loss from baseline and stratified it as > 10% and 5–10% or studies that computed weight variability were pooled using random effects model. Study quality was evaluated using the Newcastle–Ottawa Scale.Thirty eligible studies were included in the systematic review and 13 of these were included in the meta-analysis. Large weight loss (> 10%) was associated with increased risk of all-cause mortality (pooled hazard ratio (HR) 2.27, 95% CI 1.51–3.42), composite of major CV events  and CV mortality  among T2DM patients. Moderate weight loss showed no significant association with all-cause mortality  or CV outcomes . Weight variability was associated with high hazard of all-cause mortality .Large weight loss and large fluctuations in weight are potential markers of increased risk of mortality and CV events in T2DM patients. Maintaining a stable weight may have positive impact in these patients.The online version contains supplementary material available at 10.1186/s12933-022-01503-x. Weight management is strongly promoted for overweight or obese patients with type 2 diabetes (T2DM) by current guidelines to reduce the risk of diabetes complications and improve outcomes . SubstanHowever, multiple studies –12 have A previous systematic review of studies published before July 2019 found that weight gain is associated with increased risk of cardiovascular disease (CVD) and mortality, whereas the effect of weight loss on mortality and CV outcomes was not clear . We perfThis study was conducted in accordance with the PRISMA  statement. It was registered with PROSPERO (Number CRD42022301756).A comprehensive literature search was performed in the PubMed, Embase, and Cochrane Library databases to identify all relevant articles published from January 2000 to February, 2022. During the revision process, we re-searched the databases using a modified search strategy. The reference lists of the included studies and relevant review articles were manually screened to identify other potential studies.The search strategy employed a combination of Mesh and text words using the following three sets of terms: 1) “diabetes mellitus”; (2) “weight loss”, “weight change”, or “weight variability”; and (3) outcome-related terms, such as: “mortality”, “cardiovascular events”. Customised strategies for different databases were adapted where necessary. Details of the search strategy are presented in the Additional file  “diabeteThe inclusion criteria for screened full-text articles were: (1) observational cohort studies or post hoc analyses of clinical trials of adult patients with T2DM (aged ≥ 18); (2) investigated the association of weight loss or weight variability with long-term outcomes; and (3) adjusted hazard ratios (HR) or relative risks (RR) with 95% confidence intervals were reported for all-cause mortality, CV mortality and a composite of major CV events. Two reviewers (KS and YR) independently assessed the eligibility of the studies that qualified the inclusion criteria. Disagreements, if any, were resolved by consensus. If two or more studies based on the same study cohort reported data for different outcomes, they were included and analysed for specific outcomes.Two reviewers (WFY and WLQ) independently extracted the data pertaining to the following variables: first author, year of publication, original dataset, region/country, study type, year of enrolment, number of participants and proportion of female subjects, age, diabetes duration, baseline CVD exclusion, intervention, assessment of body weight, weight change interval, follow-up duration, and the study outcomes.Two reviewers (JW and YL) independently evaluated the quality of included studies by using the Newcastle–Ottawa scale. Disagreements were resolved by discussion and further review. The quality of the studies was graded as poor (< 4 points), fair (4–6 points), or good (> 7 points).2 statistics was used to assess heterogeneity. An I2 value of > 50% was considered indicative of statistically significant heterogeneity. All statistical analyses were conducted using STATA version 15.0 .The primary outcome of interest was all-cause mortality. The secondary outcome was a composite of major CV events and CV mortality. Since we aimed to assess the effect of weight loss achieved without behavioural intervention on the outcomes, for studies that included behavioural intervention, only data pertaining to the control group was pooled. Weight loss more than 10% from baseline was defined as major or large weight loss. Weight loss of 5–10% was defined as moderate weight loss. Studies with unavailable data on a specific endpoint were excluded from the pooled analysis for that endpoint. We collected available HRs or RRs after full adjustment as originally reported in each study. The list of confounding variables that were adjusted for in each study is provided in the Additional file A schematic illustration of the literature search process and study selection is shown in Fig. Characteristics of studies included in this systematic review and meta-analysis are presented in Table The studies investigated the associations of weight change with mortality and CV events using various parameters  and different cutoff levels to categorise the degree of weight change. We pooled the outcomes of studies using cutoffs of 5–10% and > 10% for weight loss. Seven studies , 36, 37 According to the Newcastle–Ottawa quality assessment scale, 20 studies were graded as having good quality and 10 studies were graded as having fair quality. Details of quality evaluation are listed in Additional file Among the 18 studies that investigated the association between weight loss and risk of all-cause mortality . There was no significant association between moderate weight loss (5–10%) and risk of all-cause mortality .Six studies –12, 19 wSensitivity analysis revealed stability of the primary results after exclusion of any one of the included studies  and CV mortality . There was no significant association of moderate weight loss (5–10%) with composite of CV events  or CV mortality  .Six studies , 22, 29 P-for-interaction > 0.05 for all). In the study by Wedick et al. [Three studies , 18, 29 k et al. , weight k et al.  [Four studies , 19, 20  for all), 19, 29.In 6 studies , 33, 36 The major findings of this systematic review and meta-analysis are as follows. First, synthesis of observational studies revealed the association of large weight loss (> 10%) with increased risk of all-cause mortality, a composite of major CV events and CV mortality. Second, moderate weight loss (5–10%) was not associated with all-cause mortality or CV events. In addition, large fluctuation in body weight was also associated with increased risk of all-cause mortality.Despite the significant heterogeneity among the studies that investigated the effect of weight loss by intentionality, the consensus was that unintentional weight loss is markedly related to higher death rate. We were unable to determine whether the weight loss was intentional or unintentional in most of the studies. However, in a majority of the included studies, behavioral interventions were not administered during the trials. And we only incorporated the data pertaining to the control group that received diabetes support and education in the Look AHEAD study  into theUnintentional weight loss may be idiopathic or occur due to various factors, such as cancer, drug interactions, depression, or other undiagnosed illnesses . Major u2, whereas a null association was observed among subjects with BMI ≥ 30 kg/m2 [Being overweight or obese is an established risk factor for cardiometabolic diseases. Weight management is highly recommended for these individuals. However, not only was weight loss found to be associated with increased mortality and CV outcomes, but several studies also reported a lower hazard of these major events in overweight or obese T2DM patients. In addition, patients who were underweight or had normal weight at the baseline had an increased risk of subsequent events. This phenomenon is referred to as the “obesity paradox”. In the ADDITION-Europe trial, large weight loss was associated with mortality among subjects with BMI < 30 kg/m30 kg/m2 . Another30 kg/m2 . However30 kg/m2 , 9, 19 dFor studies that included patients with established history of diabetes, the results were more consistent in terms of the relationship between weight loss and mortality. We analysed the results of studies that included only newly-diagnosed diabetes. Most of these studies , 23, 27 The results for the CV outcomes are more heterogeneous. In the Look AHEAD trial, participants who achieved ≥ 10% weight loss had 20% lower 10-year hazard of CVD. Since this study included only overweight or obese participants and half of them had intensive lifestyle intervention, the reduced hazard of CVD may have been partly attributable to the improvement of metabolic factors. This was demonstrated by another post-hoc analysis of this cohort . Weight In the ADDITION trial, moderate weight loss at 1 year after diabetes diagnosis was associated with reduced hazard of 10-year CVD . HoweverSince CV death is the most common cause of death in diabetic patients, the baseline CV risk profile of participants is likely to influence the association between weight loss and outcomes. The majority of these studies included participants with established CV risk factors. In the EXAMINE trial that included T2DM patients with a recent acute coronary syndrome, > 5% loss of weight was correlated with the increases in risk of subsequent CV events and mortality. Sensitivity analysis performed by excluding the EXAMINE trial yielded similar results. And within individual studies, similar adverse association with mortality was observed after adjusting for baseline CVD risk factors or after excluding participants with a history of CVD.2 was increased [All five studies that investigated the association of weight variability with outcomes found an increased HR, even though they employed different indices for the measurement of weight fluctuation. The findings of the ADDITION-Europe trial are in line with these studies . Among pncreased .Some limitations of this systematic review and meta-analysis should be considered while interpreting the results. First, there was considerable heterogeneity among the studies included in the meta-analysis. Several factors that may have contributed to the heterogeneity, including weight loss duration, duration of diabetes, severity of diabetes and underlying CVD risk. We performed sensitivity analysis and subgroup analysis aiming to reduce heterogeneity. However, the heterogeneity was primarily attributable to each of the individual study’s characteristics that could not be reduced effectively in our analyses. Therefore, we used the random effects model for meta-analysis, as this approach takes into account heterogeneity when computing the pooled effect estimate. Second, there was a difference in baseline age or initial BMI across the groups of weight change in most of the studies, which may have confounded the association between weight loss and outcomes. But almost all studies adjusted for baseline age and BMI when computing hazard ratio or relative risk ratio. And we only collected and pooled the rate ratios after full adjustment. Third, as we only pooled data of studies that categorized weight loss as 10% and 5%–10%, there were limited number of studies for the meta-analysis of the relation between weight loss and outcomes stratified by the magnitude of weight loss. Therefore, the results need to be interpreted with caution. Finally, most of the studies were observational or post-hoc analyses of clinical trials. Although major weight loss observed in most of studies was indicative to be unintentional, the effect of intentional weight loss needs to be evaluated in long-term intervention trials.Large weight loss and large fluctuations in weight are potential markers of increased risk of mortality and CV events in T2DM patients. Weight change as a global variable for physical condition may reflect the overall effect of multiple pathophysiologic processes, indicating disease progression and worsening health status. Management for T2DM patients should not focus solely on weight loss. Establishing healthy lifestyle and maintaining a stable weight may be more important.Additional file 1. Details of the search strategy for different databases.Additional file 2. Additional Tables S1–S6 and Figures S1–S2.


Assessing circadian rhythmicity from infrequently sampled data is challenging; however, these types of data are often encountered when measuring circadian transcripts in hospitalized patients.We present ClinCirc. This method combines 2 existing mathematical methods (Lomb-Scargle periodogram and cosinor) sequentially and is designed to measure circadian oscillations from infrequently sampled clinical data. The accuracy of this method was compared against 9 other methods using simulated and frequently sampled biological data. ClinCirc was then evaluated in 13 intensive care unit (ICU) patients as well as in a separate cohort of 29 kidney-transplant recipients. Finally, the consequences of circadian alterations were investigated in a retrospective cohort of 726 kidney-transplant recipients.ClinCirc had comparable performance to existing methods for analyzing simulated data or clock transcript expression of healthy volunteers. It had improved accuracy compared with the cosinor method in evaluating circadian parameters in PER2:luc cell lines. In ICU patients, it was the only method investigated to suggest that loss of circadian oscillations in the peripheral oscillator was associated with inflammation, a feature widely reported in animal models. Additionally, ClinCirc was able to detect other circadian alterations, including a phase shift following kidney transplantation that was associated with the administration of glucocorticoids. This phase shift could explain why a significant complication of kidney transplantation (delayed graft dysfunction) oscillates according to the time of day kidney transplantation is performed.ClinCirc analysis of the peripheral oscillator reveals important clinical associations in hospitalized patients.UK Research and Innovation (UKRI), National Institute of Health Research (NIHR), Engineering and Physical Sciences Research Council (EPSRC), National Institute on Academic Anaesthesia (NIAA), Asthma+Lung UK, Kidneys for Life.  Circadian biology has profound effects on both outcome and pathophysiology  in many It is possible to measure circadian outputs in order to assess circadian oscillations. However, these outputs may be influenced by noncircadian mechanisms , 8, caus2 periodogram, mFourFit, maximum entropy spectral analysis, and spectrum resampling) are potentially unreliable, as they rely on collection periods lasting several days  at once or make untested assumptions about the parameters, such as assuming that the period is approximately 24 hours. Therefore, existing single-step circadian methods . On simulated data and healthy volunteers, ClinCirc had comparable sensitivity and specificity when compared with existing methods. In the critical care setting, ClinCirc was the only method tested that revealed that loss of circadian oscillations was associated with inflammation, a feature widely reported in animal models –12. FurtTo evaluate the optimal mathematical method for circadian analysis in patients, simulated data were analyzed with 9 existing mathematical techniques. All data had a sampling frequency of 4 hours and were sampled over a period of 24 to 48 hours, replicating the constraints of clinical circadian analysis. In addition to the 9 existing mathematical techniques, we evaluated ClinCirc, a method that combined L-SP with cosinor analysis. In the preprocessing step, L-SP was used to identify the presence of a circadian oscillation by defining the dominant harmonic. An unconstrained cosinor analysis was then used as further preprocessing to exclude cases with excessively high or low periods based on the fit to a single sinusoid. To determine the amplitude and phase of any detected circadian waveform, a cosinor analysis was then used where the period was constrained from 18 to 30 hours .https://doi.org/10.1172/JCI162775DS1). The exceptions were GPR (Matérn), which had sensitivity of 99.8% over 48 hours, GPR (periodic), which had a sensitivity of 0% over 24 hours or 48 hours, and some of the constituent parts of MetaCycle. The addition of random noise , which had a sensitivity of 41%. GPR (periodic) had a sensitivity of 34.6%, with the remaining methods all having a sensitivity below this  with a further 7 being sampled 48 to 72 hours after their surgery.The clinical applicability of ClinCirc was then investigated by analyzing the expression of clock transcripts making up the molecular oscillator in peripheral blood from 2 different patient cohorts. The first cohort of patients were critically ill patients admitted to the intensive care unit (ICU) . These iP < 0.05, χ2 test; Initially, the presence or absence of detected circadian oscillations of individual peripheral oscillator transcripts was quantified in patient cohorts. The proportion of ICU patients in whom ClinCirc detected a circadian oscillation was reduced for NR1D1 and PER2 compared with that in healthy volunteers   oscillated in a circadian manner in over 50% of ICU patients with standard detection . For 3 oχ2 test) . The preχ2 test) .2r = 0.33–0.56, The association between inflammatory mediator expression and molecular oscillator circadian oscillations was then examined, as this has been previously reported in animal models . ICU patP < 0.01, ANOVA, post hoc Tukey’s test; 2r = 0.588, Pearson’s; Pentraxin-3, an acute-phase protein, had the greatest fold change of all the measured inflammatory mediators among ICU patient groups. However, this acute-phase protein is not routinely measured in the ICU. Therefore, the expression of an acute-phase protein that is routinely measured (C-reactive protein [CRP]) was also investigated. CRP was 4-fold higher in patients in whom the circadian oscillations in the molecular oscillator were reduced compared with clinical populations in whom the detection of circadian oscillations was normal (ICU or kidney-transplant recipients)  and also phase (±0.2 hours) estimation  comparedP < 0.01, 2-way ANOVA post hoc Dunnett’s; P = 0.01–0.02, 2-way ANOVA post hoc Dunnett’s; Twenty-four hours after kidney transplantation, the amplitude of circadian oscillations was increased compared with that of healthy volunteers for NR1D1, PER1, PER2, PER3, and CRY2 , the mean phase for 4 clock genes  was significantly shifted compared with that of healthy volunteers or kidney-transplant recipients 72 hours after kidney transplantation . In cont hours, t2r = 0.9, P < 0.01; 2r = 0.9, P < 0.01;Figure 3D). Similar linear correlations were observed for the remaining 8 clock genes’ acrophases, suggesting that the molecular oscillator had been entrained to allograft reperfusion time . Since the incidence of DGF is affected by the donor, the cohort was split according to whether the donor had experienced brain  or circulatory  death. The probability of DGF from DCD donors did not show a relationship with organ-reperfusion or organ-harvest time   .n = 43), with cosinor detecting 54 waveforms and GPR (Matérn) detecting 63 circadian waveforms. The goodness of fit (2r) and leverage distance (distance between maximum outlier and predicted oscillation) were then compared between methods. Initially, the performance of ClinCirc was compared with GPR (Matérn) using waveforms that were only described as circadian by one method and not the other. The adjusted 2r was higher for waveforms that ClinCirc deemed circadian but the GPR (Matérn) method did not  defined as circadian for the whole clinical cohort was then compared . ClinCir did not . In cont did not . Then th did not  and the  did not .2r). Therefore, ClinCirc analysis of circadian oscillations in the molecular oscillator provides translationally important insights into circadian biology in clinical situations in which it is difficult to detect circadian oscillations.ClinCirc analysis of the peripheral oscillator revealed that inflammation is associated with reduced rhythmicity in ICU patients. In the context of kidney transplantation, in which inflammation is suppressed by medication, ClinCirc revealed that the molecular oscillator was shifted and associated with allograft reperfusion time. Although two other methods  also revealed similar findings in kidney-transplant patients, the strongest association was observed with ClinCirc. In the ICU cohort, ClinCirc was the only method out of the 10 tested to reveal an association between rhythmicity and inflammation that has previously been reported in animal models . One pos2r value. The resulting poor specificity could explain why this and previous studies have not observed an association between inflammation and circadian oscillations  method were lower than for ClinCirc, again suggesting that the lack of specificity could hide clinically important associations. Further optimization of kernels is likely to improve this performance; however, GPR techniques are also quite computationally intensive and so would require more resources than ClinCirc to run.Measuring circadian oscillations in patients is challenging, due to practical considerations that limit the length of the sampling period as well as the number of samples that can be collected. Our data would suggest that the widely used cosinor approach is prone to overfitting in this scenario, which was demonstrated by an increased leverage distance compared with ClinCirc alongside a lower llations  using thllations  kernel uP value, e.g., using CirCaN  and were likely to survive 48 hours, as identified by the admitting ICU physician, were considered for entry between September 2017 and September 2019 if they were aged over 18. Patients were excluded a priori if they had BMIs over 35, nonreversible organ dysfunction, psychiatric illness requiring inpatient treatment within the last 12 months, a history of shift work, a medically treated sleep disorder (including use of continuous positive airway pressure [CPAP]), end of life care, alcohol intake greater than 70 units per week in last year, nonreversible end-stage renal failure, a cardiac arrest in the last year, cancer treatment or surgery in the last 30 days, suspicion of a highly transmissible infection, were pregnant or breastfeeding, or had a permanent neurological deficit due to the potential effects this could have on circadian oscillations. Inclusion and exclusion criteria were set a priori based on discussion between the investigators and a review of the literature. Diagnosis and clinical characteristics of the patients were independently verified by review of the charts by two General Medical Council–registered doctors and are listed in the patient demographics. The number of cases during the sampling period determined the sample size. The sample size used to evaluate alterations to the peripheral oscillator was comparable to those in other studies with similar objectives , 32–35. Patients undergoing cadaveric kidney transplantation at Manchester Foundation NHS trust between 2015 and 2019 were considered for recruitment. Consent was provided before transplantation when the retrieval team was retrieving the organ. Following consent, blood was taken every 4 hours for the first 24 hours after the operation (24 hours) or between 48 and 72 hours after the operation (72 hours). Patients were excluded from the study if the surgeon thought they had a high probability of needing hemodialysis before or during the circadian sampling period. Clinical parameters, e.g., CRP, were recorded from the clinical chart over the sampling period.The data for DGF were taken from a 10-year retrospective service evaluation starting when the transplant protocols were modified in 2007 at MFT. Patients were identified following analysis of the transplant center’s database. The presence of delayed graft failure was then identified following review of the patient’s health-care record. DGF was defined for this study as the need for dialysis within the first 7 days after transplantation. The time of organ harvest and organ reperfusion as well as the type of cadaveric transplant were also obtained from the patient’s health-care record.Blood (3 ml) was collected in Tempus Blood RNA tubes for RNA analysis. Each tube was inverted 3 times, lysing the blood, and then the tube was frozen (–20°C) until analyzed. RNA was then extracted from the sample using Tempus Spin RNA Isolation Reagents following the manufacturer’s instructions and subsequently converted into cDNA using an Applied Biosystems High Capacity RNA-to-cDNA kit. Quantitative PCR (qPCR) was performed using prevalidated PrimePCR TaqMan primer assays from Bio-Rad, using TBP as the housekeeping gene.g for 10 minutes, the plasma was aliquoted and stored at -80°C until further analysis. The protein concentration of inflammatory mediators was analyzed using the Pro Human Inflammation 37-plex Bio-Plex per the manufacturer’s instructions (Bio-Rad).Blood (6 ml) was drawn into Vacutainer Lithium Heparin Tubes (BD). Following centrifugation at 1,500Two studies were analyzed for control data. For the first study , the dathttps://figshare.com/s/700cedb00253847212ae along with instructions, which are available at https://figshare.com/s/d492ca6f244380f8e650 An example data set can be found at https://figshare.com/s/7a8d545272046bf53f22The ClinCirc algorithm is available at t tests, χ2 test) or Mathematica (Watson 2U test) to compare phases. GPR (periodic) was implemented using custom routines written in MATLAB. GPR (Matérn) was implemented using previously published routines  as well as the institution (Manchester University NHS Foundation Trust). Written, informed consent was obtained prior to patient enrollment. Consent was provided by patients if they were deemed to have capacity; otherwise, assent was provided by a professional or close relative, which was subsequently confirmed by the patient when they regained capacity.JFB, GK, and PSC conceived the study, analyzed the data, and wrote the manuscript. CJ, TH, OEA, and ALH developed and applied the mathematical methods and helped write the manuscript. SP and TS helped acquire data and critically appraised the manuscript. DVD, JG, TF, AW, JBS, MKR, TA, and PD all helped with the design of the study and study analysis and provided critical feedback on the manuscript.


Furcellaria lumbricalis is a potential candidate for biomedical applications due to its gelation properties and mechanical strength. In the present study, immobilization of furcellaran onto polyethylene terephthalate surfaces by a multistep approach was studied. In this approach, N-allylmethylamine was grafted onto a functionalized polyethylene terephthalate (PET) surface via air plasma treatment. Furcellaran, as a bioactive agent, was anchored on such substrates. Surface characteristics were measured by means of contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Subsequently, samples were subjected to selected cell interaction assays, such as antibacterial activity, anticoagulant activity, fibroblasts and stem cell cytocompatibility, to investigate the Furcellaran potential in biomedical applications. Based on these results, furcellaran-coated PET films showed significantly improved embryonic stem cell (ESC) proliferation compared to the initial untreated material.Surface coatings of materials by polysaccharide polymers are an acknowledged strategy to modulate interfacial biocompatibility. Polysaccharides from various algal species represent an attractive source of structurally diverse compounds that have found application in the biomedical field. Furcellaran obtained from the red algae  Furcellaria lumbricalis. It is negatively charged and composed of units consisting of a fragment (1→3) β-D-galactopyranose with a sulfate group at C-4 and (1→4)-3,6-anhydro-α-D-galactopyranose. Structurally, furcellaran resembles the algal polysaccharide κ-carrageenan, with a major structural difference that only approximately 60% of the D-galactose units are 4-sulfated [Polysaccharides, in general, have increasingly attracted attention owing to their excellent antitumor, antioxidant, antibacterial, anti-inflammatory and anticoagulant activities . Moreovesulfated . The numsulfated . Higher sulfated  and is psulfated  and β-lasulfated , and fibsulfated . The intsulfated ,13. The sulfated ,15. The sulfated , essentisulfated  and nanosulfated .Polymeric biomaterials that are intended for clinical use must have excellent mechanical properties, as well as adequate surface properties to ensure biocompatibility while interacting with living tissue. The current problem that can be encountered with biomaterials used for implants and other devices may be acute and chronic inflammatory responses, ultimately leading to fibrous capsule formation and thereby impairing normal tissue growth ,20. FurtTherefore, activation procedures have been carried out to make the polymer surface more hydrophilic and obtain specific features by introducing functional groups  for further covalent bonding with selected agents . The typThe plasma-treated polymer results in a negatively charged surface; therefore, subsequent immobilization of a potential anionic polysaccharide is challenging. The negative charge prevents the formation of covalent bonds between the molecule interfaces and polymer surface embedded radicals due to electrostatic repulsive forces. The binding affinity can be increased by reducing the negative charge of the polymer surface. One promising solution might be introducing more positively charged groups through mediators, such as N-allylmethylamine (MAAM). This liquid chemical is able to graft onto a plasma-treated polymer surface by a copolymerization process to create a polymer brush structure with a high density of positively charged amino groups via a radical “surface from” reaction in the gaseous phase ,33. SuchIn the present study, poly (PET) was used as a substrate due to its unique mechanical properties, biostability and its moderate inflammatory response. The Biostability of PET is mainly due to the presence of hydrophobic aromatic groups with high crystallinity which restricts hydrolytic breakdown. The low degradability of the PET offers enduring support over time along with ultimate performance during the patient’s lifetime. This can also surmount the problems of asynchronous degradation with new tissue regeneration and harmful end products of degraded polymers . Hence, This research is the first study dedicated to the description of various furcellaran applications in biomedicine. Moreover, to the best of our knowledge, no study using furcellaran as a polysaccharide film layer for PET functionalization has been reported. The objective of this study is to represent a method for immobilizing furcellaran onto PET surfaces. For this purpose, RF plasma discharge is applied onto the PET surface to create oxidative functional groups for further covalent binding with selected agents. First, MAAM is grafted onto plasma-treated PET to create a high-density polymer brush for improved adhesion interaction properties. Consequently, furcellaran immobilization on such a treated surface is performed. As-prepared substrates are subjected to various selected cell interactions. Namely, the antibacterial activity, anticoagulant activity, fibroblasts and stem cell cytocompatibility of the samples were investigated.w/v) furcellaran and κ-carrageenan in distilled water. Diiodomethane  and formamide  were supplied by Sigma–Aldrich .Polyethylene terephthalate (PET) in rectangle form sheets (55 × 30 mm) was rinsed with distilled water and subsequently dried at room temperature. Monomer N-allylmethylamine (MAAM) was purchased from Sigma–Aldrich . κ-carrageenan (κ-CA) and furcellaran (FUR) of different water gel strengths  were obtained from Est-Agar AS . The solution was prepared by dissolving 0.1%  for 60 s at a radio frequency of 13.56 MHz. The discharge matching power was set to 50 W, and the applied air feed rate was 20 sccm (standard cubic centimeter per minute). The pressure inside the chamber was approximately 60 Pa. Some of the plasma-treated PET samples were subsequently immediately exposed to saturated MAAM vapors to achieve radical graft monomer polymerization towards polymer brushes, which are suitable for biological agent immobilization (including polysaccharides). For comparison, the characterization and ensuing treatments of PET samples were also performed without previous MAAM grafting .w/v) solution of κ-CA and FUR for 24 h at room temperature for their immobilization onto a previously prepared polymer brush. After deposition, the samples were removed from the solution and washed in distilled water to remove residual unbound polysaccharide content. Finally, the samples with immobilized polysaccharide layers were dried overnight at room temperature.MAAM grafted and nongrafted PET samples were immersed into a 0.1% . Lewis acid-base parameters of the surface free energy of the samples were evaluated from contact angle values using appropriate testing liquids, i.e., water, diiodomethane, formamide . Five seDroplets of testing liquid with a volume of 5 µL were placed onto each sample and captured by a CCD camera system. This droplet volume was set for several reasons. The most important factor was the fact that larger drops are deformed by gravity. Smaller drops give results with higher measurement errors.The chemical composition was analyzed with X-ray photoelectron spectroscopy (XPS) using TFA  with MultiPak software to determine the concentration of elements. The samples were irradiated with X-rays with a 400 µm spot size generated with monochromatic Al Kα1,2 radiation at 1486.6 eV. The emitted photoelectrons were detected with a hemispherical analyzer placed at an angle of 45° relative to the normal plane of the sample surfaces.The surface morphology of each sample was monitored by a NANOSEM 450  scanning electron microscope operated at 5 kV with an Everhart–Thomley detector (ETD). The images were obtained at a magnification of 20,000×. The samples were coated with Au/Pt prior to observation.Escherichia coli (CCM 4517) and Staphylococcus aureus (CCM 2022) were prepared in 1/500 nutrient broth and diluted to obtain a certain bacterial concentration between 2.5 × 105 cells/ml and 10 × 105 cells/ml. Thus, the prepared solutions were used as the test inoculum, which was dispensed onto each sample (25 × 25 mm) in a volume of 0.4 mL. Subsequently, the samples were covered with ethanol-disinfected polypropylene foil (PP) (20 × 20 mm2) and incubated at 35 °C and 95% relative humidity for 24 h. After the incubation period, each sample with PP films was washed with 10 mL of SCDLP broth  to determine the recovery rate of the bacteria. The recovered bacterial suspensions were subjected to 10-fold serial dilutions. The viable bacteria count was evaluated by the pour plate culture method after 24 h of incubation.The antibacterial activity was determined according to a modified version of the ISO 22196 standard for investigation of the antibacterial effect on modified plastic materials. First, all samples were sterilized by UV radiation and then placed in sterile Petri dishes. Standardized bacterial suspensions of Blood was obtained by venous puncture from a healthy donor in accordance with the Helsinki Declaration and placed into blood collection tubes  covered by prepared PET samples. The obtained human blood plasma was treated with 3.2% citric acid (109 mmol/L) and then centrifuged at room temperature for 15 min at 3000 min. The anticoagulant activity was determined by means of prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (aPTT) using a SYSMEX CA-1500  instrument. Each of the samples was examined three times.−1 penicillin/streptomycin  was used as the culture medium. The cells were seeded onto samples at a concentration of 2 × 104 cells per mm2 and incubated at 37 ± 1 °C for 72 h. After 72 h of cell culture, the cell viability was evaluated using the 3--2,5-diphenyltetrazolium bromide (MTT) assay . First, the culture medium was removed, and 100 µL of growth medium containing MTT dye solution (5 mg/mL in PBS) was added to the cultures. Then, the cells were incubated at 37 °C in a humidified atmosphere for 4 h. Following the removal of the growth medium, DMSO  was added to dissolve the formed formazan crystals on the sample surface. The absorbance was measured using an Infinite M200 Pro NanoQuant absorbance reader  at wavelengths of 570 nm (test) and 690 nm (reference). The reported values are the means of three replicates and are expressed as percentages of the control value.The cytotoxicity was tested using a mouse embryonic fibroblast cell line  according to the EN ISO 10993-5 standard with some modifications. The tested samples with dimensions of 10 × 10 mm were exposed to UV radiation  for sterilization. ATCC-formulated Dulbecco’s modified Eagle’s medium  containing 10% calf serum  and 100 U mL2 in humidified air. After 120 h (5 days) of proliferation, the cells were fixed and stained to visualize the DNA with Hoechst 33258  and observed by fluorescence microscopy using an Olympus IX 81 inverted phase-contrast microscope . An MTT proliferation test was performed. All tests were performed in four separate sets. Statistical significance was determined by ANOVA with post hoc Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001The embryonic stem cell line ES R1 was propagated in an undifferentiated state by culturing on two series of prepared samples. One series was coated with 0.1% gelatine, and the other was not. Dulbecco’s Modified Eagle’s Medium with high glucose, pyruvate, 16.5% calf serum , 1% penicillin/streptomycin , 100 mM nonessential amino acids , 0.05 mM b-mercaptoethanol , and leukemia inhibitory factor  at a concentration of 5 ng.mL-1 was used as the culture medium. First, both sides of the prepared samples were sterilized by UV light for 30 min. Then, the cell suspension was seeded on the tested samples at a concentration of 20,000 cells per mL and incubated at 37 °C in 5% COs of untreated PET foil, low values were calculated due to its low surface wettability. The value of γs after air plasma exposure seemed to be distinctly higher, indicating that the surface had a relatively significant polarity. Variations between the γs values of plasma-treated PET and immobilized PET were not significant. The results indicate successful immobilization of FUR and κ-CAR onto the PET surface, as subsequently demonstrated in detail by XPS.Wettability changes resulting from surface modifications were determined from contact angle data for various testing liquids and are shown in The prepared samples with the different surface treatments were investigated by the XPS method, and their elemental compositions are shown in The surface morphology was examined by means of SEM performed with an Everhart–Thomley detector without a conductive coating of the samples and represented at a 10 μm resolution. The results are shown in Staphylococcus aureus and Escherichia coli strains were evaluated by the number of viable cells calculated in the agar plates after 24 h of incubation. As shown in The antibacterial activity levels of PET films against Along with the presence of the sulfate group, the antibacterial activity of polysaccharides can be enhanced via chemical modification by incorporating other functional groups, such as carboxylic and carboxymethyl groups, aldehyde, amine, alkylamine, and quaternary ammonium groups. The hydroxyl groups can be also grafted with organic moieties offering extra functional groups to improve bioactivities according to the given application ,48. BaseThe anticoagulant activity of PET samples was evaluated by thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (aPTT) coagulation assays, and the obtained results are summarized in In general, the blood coagulation system is divided into two initiating pathways: the tissue factor (extrinsic) pathway and the contact factor (intrinsic) pathway. These pathways meet in a final common pathway where factor Xa converts prothrombin to thrombin, which then cleaves fibrinogen to fibrin monomers.PT is a functional measure of the extrinsic and common pathways, and the physiological value is in the range of 11–13.5 s. As seen, all samples are within the range, meaning that the extrinsic pathway of coagulation was not inhibited by any of the tested samples. These data are consistent with a study on the anticoagulant activity of major types of carrageenans .aPPT is a functional measure of the intrinsic pathway as well as the common pathway, and the reference range is between 22 and 33 s. As the results show, the clotting times of samples PET_DC_8500 and PET_DC_KAPA were slightly prolonged, and therefore, interfered with the intrinsic coagulation process.TT is a measure of functional fibrinogen in plasma, and for healthy donors, the clotting time range is between 12.5 and 19 s. PET_DC_KAPA displayed the highest value of clotting time and thereby mildly exceeded the threshold for anticoagulant activity. As follows, plasma-treated PET exhibited a prolonged time over the threshold. The hydrophilic surface achieved by plasma treatment may be associated with lower protein adsorption and the activation of blood components. Another factor influencing protein adsorption on hydrophilic surfaces is the type of charge. Negatively charged surfaces typically lead to a lower extent of protein adsorption than positively charged surfaces because many proteins have a net negative charge at physiological pH values . These rThus, higher values were observed in plasma-treated PET and polysaccharide-immobilized samples in combination with plasma treatment, which correlated with the content of sulfates and hydrophilic surfaces ,48,49,50The cytotoxicity of individual samples was evaluated using a mouse embryonic fibroblast cell line (NIH/3T3) according to the ISO 10993-5 standard. Cytotoxicity tests are the first option when the biocompatibility of materials, including polymers, is assessed. Measurements were performed in triplicate with untreated PET as a reference. Error bars represent standard deviations. 2) functional groups and reveal the optimum polymer brush for the immobilization of the tested polysaccharides. Amine groups are known to promote cell adhesion because of their positive charges, which can attract negatively charged biomolecules, such as proteins under physiological conditions. It was expected that MAAM-treated samples would demonstrate increased cell proliferation due to a higher quantity of polysaccharide agents immobilized. As shown in Examples of versatile, non-animal derived and inexpensive materials that are able to support the proliferation of ESCs are limited. Many of the new biomaterials used to develop stem cell microenvironments use biopolymers adsorbed or covalently immobilized to the surface to improve the biocompatibility of synthetic polymers . FurcellIn this work, furcellaran deposition onto a PET surface via RF air plasma discharge activation followed by grafting of a MAAM monomer was studied. The successful immobilization of polysaccharides onto grafted and nongrafted MAAM monomer brushes was confirmed by the contact angle and XPS results, where the surface wettability of the resultant films increased compared to the PET reference, and the sulfur content characteristic of the tested polysaccharides was detected. SEM images of coated PET samples demonstrated a rather microtextured homogenously distributed layer, which had positive effects on the uniform adhesion and proliferation of fibroblasts and thus exhibited nontoxicity for all functionalized samples. All samples had minor effects on the antibacterial activity and blood coagulation, as indicated by the clotting times of APPT, PT and TT. However, MAAM nongrafted samples of immobilized FUR significantly increased ESC proliferation, in contrast to the MAAM grafted ones. The low ESC proliferation rate for MAAM grafted samples might be due to the insufficient interaction of polysaccharides with amine groups. The obtained data for furcellaran were compared with the results for κ-carrageenan. These preliminary results show that furcellaran itself has significant biomedical application prospects and paves the way for future investigations.


Montipora capitata, one of the main reef-building coral species in Hawai'i, is known to host a diversity of symbionts, but it remains unclear how they change spatially and whether environmental factors drive those changes. Here, we surveyed the Symbiodiniaceae community in 600 M. capitata colonies from 30 sites across Kāne'ohe Bay and tested for host specificity and environmental gradients driving spatial patterns of algal symbiont distribution. We found that the Symbiodiniaceae community differed markedly across sites, with M. capitata in the most open-ocean (northern) site hosting few or none of the genus Durusdinium, whereas individuals at other sites had a mix of Durusdinium and Cladocopium. Our study shows that the algal symbiont community composition responds to fine-scale differences in environmental gradients; depth and temperature variability were the most significant predictor of Symbiodiniaceae community, although environmental factors measured in the study explained only about 20% of observed variation. Identifying and mapping Symbiodiniaceae community distribution at multiple scales is an important step in advancing our understanding of algal symbiont diversity, distribution and evolution and the potential responses of corals to future environmental change.The survival of most reef-building corals is dependent upon a symbiosis between the coral and the community of Symbiodiniaceae.  The s Pacific . Cladocose genus ,19 whileiability  and is aiability ,19–22.Durusdinium often comes at an energetic cost, as it decreases the growth and metabolite exchange rate of the host [Thermal stress is the main threat affecting corals worldwide –27. Sea the host –52.Montipora capitata, which is one of the most abundant corals in Kāne‘ohe Bay and may harbour a community of Cladocopium (C), Durusdinium (D) or both symbiont genera [Durusdinium glynnii  to identify the Symbiodiniaceae assemblage and the local-scale environmental drivers of symbiont community composition for 600 colonies of the common reef-building coral . 2. 2.1et al. [M. capitata were tagged, for a total of 600 tagged colonies distributed across 30 sites spread equally across the five hydrodynamically defined blocks. In early 2018, about 1 cm2 was sampled from each of 600 tagged Montipora capitata colonies. Sampled fragments were immediately preserved in 70% ethanol and stored at −20°C until processed. DNA was extracted using the Nucleospin Tissue Kits  following manufacturer instructions and quantified by fluorimetry . Only corals that appeared healthy were sampled for this study.Details of the stratified random sampling design and environmental data are detailed by Caruso et al. . Brieflyet al. ,65 and set al. . Within Data loggers  recorded temperature and sediment traps were collected regularly from each site  and used. 2.2et al. [Symbiodiniaceae amplicon library preparation and sequencing followed the protocol outlined in . Brieflyet al.  to inclu. 2.3ITS2 sequence variance .Raw sequences were first demultiplexed using Cutadapt . DemultiMany terms have been used to describe the Symbiodiniaceae unity of resolution. To avoid ambiguity, we restrict our use to ‘Symbiodiniaceae type’ and ‘Symbiodiniaceae profile’. A type refers to Symbiodiniaceae taxa that have a specific sequence as their most abundant sequence. A Symbiodiniaceae profile is a set of ITS2 sequences that have been found in a sufficient number of independent samples to be identified as a ‘defining intragenomic variant’ (DIV). For example, C17 is a Symbiodiniaceae type and C17d/C31-C21-C17e-C21ac-C17f-C17 g is a Symbiodiniaceae profile with C17d present in higher abundance than the other types.. 2.4adonis , and pairwise.adonis (for pairwise PERMANOVA) in the vegan package [R2 from the PERMANOVA was plotted in a dendrogram using the function pheatmap in the package gplots [metaMDS was used in the R package vegan [All analyses were done in the R statistical environment . Bray-Cu package , each wie gplots . The funge vegan  to genercapscale. After running an ANOVA to check the significance of the constrains axis as well as the significance of the environmental variables , we visualized the significant variables in the dbRDA. Samples were considered to have majority Cladocopium (C) or Durusdinium (D) if the proportion of either algal symbiont exceeded 80% in the sample  using the function ied from ). All ret Zenodo .. 3. 3.1Montipora capitata samples returned high-quality reads. Prior to quality filtering, these samples included 3 285 481 sequences which were reduced to 1 632 505 following quality control and minimum entropy decomposition with SymPortal, an average of 2968 sequences per sample. 11 386 sequences had not been previously curated by SymPortal and were unique to this dataset. A total of 283 Symbiodiniacae types were identified, 85% belonging to the genus Cladocopium, and 15% belonging to the genus Durusdinium. Twenty-six ITS2 type profiles were identified across all samples, 23 of which were from the genus Cladocopium, with the remaining three belonging to the genus Durusdinium. Overall, 43% of Montipora capitata hosted Cladocopium only, 11% hosted Durusdinium only, and 46% hosted a combination of both genera. From those mixed colonies, 32.5% were dominated by Cladocopium (C greater than 80%), while 37% of colonies were dominated by Durusdinium (D > 80%). To confirm differences in read number do not change the results or interpretation, we excluded 20 samples whose number of reads were more than two standard deviations above or below the mean.550 . 3.2F25 = 6.5632, p = 0.001; electronic supplementary material, table S2). Pairwise comparisons revealed that blocks 1 and 5 were significantly different, whereas blocks 2–4 were rarely significantly different from one another .Symbiodiniaceae composition varied significantly among corals within each of the environmentally delineated blocks  which did not have any samples in which Durusdinium was detected . It is noteworthy that sites 5_3 and 5_6 were also the least diverse of all we sampled, and unlike other sites, some colonies hosted only a single type (C3) of algal symbiont . Corals within block 5 hosted significantly less Durusdinium symbionts than any other site, with all sites including corals hosting a majority of Cladocopium. Site 2_2 had the highest relative proportion of Durusdinium and site 3_2 was the only site to have three profiles of Durusdinium.Most sites had corals with both . 3.3M. capitata hosting majority Cladocopium are distinct from M. capitata hosting majority Durusdinium or mixed C & D . dbRDA showed that ed C & D . PERMANO. 4Montipora capitata. Previous surveys of the Symbiodiniaceae community structure of M. capitata across Kāne‘ohe Bay [et al. [et al. [Cladocopium and Durusdinium but did not have the resolution to identify the diversity of subtypes of Cladocopium and Durusdinium as in this study. However, it is not the additional resolution so much as the geographic range of sampling that appears to underlie the difference, because Innis et al. [et al. [et al. [et al. [Here, we contribute to a more nuanced understanding of coral algal symbiosis by examining the associations of environmental drivers across a small spatial scale (approx. 10 km) on the community composition of Symbiodiniaceae in one of the dominant reef-building corals in the region, ‘ohe Bay ,61 found [et al. ) sampleds et al.  had no s [et al.  conclude [et al.  in that Cladocopium is present, C31 is typically most abundant followed by C17 and C21, whereas for Durusdinium, D1 is the most abundant, followed by D4 and D6), no matter the location  are detected among colonies across Kāne‘ohe Bay, with C31, C17 and C1 being the most common. Similarly, we detected six types of Durusdinium  with D1, D4, and D6 being the most prevalent. These results are consistent with multiple studies reporting strong Symbiodiniaceae specificity with type C31 being the most common symbiont associated with Montipora capitata in Hawai‘i [Using a high-throughput metabarcoding approach, our study shows that the Symbiodiniaceae community composition varied at all spatial scales examined, both among the sites within each region and among hydrodynamically defined blocks across the bay. Interestingly, the relative proportion of algal symbiont types is relatively consistent among the colonies  hosted Symbiodiniaceae communities that were significantly different from blocks in the centre of the bay. We find four factors  that are significant drivers of Symbiodiniaceae community composition , although these four significant factors combined explained less than 20% of the variation in symbiont community composition. Although 80% of the variation remains unexplained, meta-analyses show that environment generally explains about 20% of the variation in community structure across terrestrial, freshwater and marine ecosystems [Despite considerable variation in the composition of the communities of Symbiodiniaceae hosted by osystems . Other fosystems , nutrienosystems ,79 and p. 4.1Durusdinium, with most colonies hosting only Cladocopium  b. 4.2Suspended sediments can both impact corals directly and alter their light penetration and irradiance. Sedimentation can also impact corals negatively through increased nutrient input, damaging the coral surface and by making it harder for corals to feed and photosynthesize –94. Sedi. 4.3Durusdinium, while block 1 and block 5  had the lowest proportion, suggesting that blocks at the extreme ends of the bay may be more vulnerable to bleaching events. This prediction is consistent with coral surveys during the 2015 bleaching event in Kāne‘ohe Bay which found the highest levels of bleaching and paling in the north (70%) and south (60%) of the bay [The role of temperature in mediating the symbiosis between the coral host and their algal endosymbionts has been widely studied ,32,95,96 the bay .Here we advance previous work by showing that algal symbiont communities within a single species of coral in a single embayment are finely tuned to their environmental conditions. Whether community response results from selection for Symbiodiniaceae types living under different environments, adaptive shuffling of Symbiodiniaceae communities in response to environmental conditions, or both, remains to be determined. Comparing microscale environmental variability ,102 to a. 5M. capitata colonies across a relatively small spatial gradient (approx. 10 km) within Kāne‘ohe Bay showed that algal symbiont community structure is associated with depth and temperature. This fine-scale variation in algal symbiont community composition across local environmental gradients suggests that algal symbiont communities can adaptively match the environmental conditions surrounding the holobiont. Previous studies of Symbiodiniaceae in the Bay focused on the three central regions that exhibit the least environmental variability among sites across the environmental gradient from north to south. Here we extend the sampling to include the full gradient across the bay and overturned previous conclusions that algal symbiont structure did not differ significantly among regions of the bay. Our results support that conclusion for symbiont communities in the central portion of the bay, but show that both environmental extremes of the far northern and southern regions of Kāne‘ohe Bay sampled here for the first time differ significantly from those in the central region. These results imply that the community composition responds to the conditions under which the holobiont is living, setting the stage for understanding the role of environmental conditions in how Symbiodiniaceae communities are distributed in space and time.Fine-scale sampling of 600 Montipora capitata. While depth was the main factor driving algal symbiont community composition in our study, several aspects of temperature  likewise appear to be drivers of algal symbiont community composition. We also note that many factors correlate with depth, such as light, temperature, sedimentation rate and water flow, such that fine-scale measurements of the full range of environmental factors surrounding individual colonies through time will be needed to pinpoint the most important environmental drivers of algal symbiont community structure. Regardless of the ultimate suite of parameters that drive algal symbiont community structure in corals, our study shows that Symbiodiniaceae communities are attuned to fine-scale environmental gradients and that understanding these complex interactions across the heterogeneous mosaic of coral reef environments is needed to better predict spatial patterns in biological responses such as bleaching susceptibility.Our study highlights the complex interactions among environmental factors and algal symbiont diversity in the reef-building coral


Nowadays, exome sequencing is a robust and cost-efficient genetic diagnostic tool already implemented in many clinical laboratories. Despite it has undoubtedly improved our diagnostic capacity and has allowed the discovery of many new Mendelian-disease genes, it only provides a molecular diagnosis in up to 25–30% of cases. Here, we comprehensively evaluate the results of a large sample set of 4974 clinical exomes performed in our laboratory over a period of 5 years, showing a global diagnostic rate of 24.62% (1391/4974). For the evaluation we establish different groups of diseases and demonstrate how the diagnostic rate is not only dependent on the analyzed group of diseases  but on the specific disorder . We also detail the most frequent mutated genes within each group of disorders and discuss, on our experience, further investigations and directions needed for the benefit of patients. Although nearly 85% of the disease causing variants are located in the coding regions or in the canonical splicing sites2, a global molecular diagnosis is nowadays only established in up to 25%-30% of cases7. However, this ES diagnostic capacity is variable and it depends on the analysed disorder7, the type of study performed (TRIO (31–37%) vs Singleton (21–22%)8 and the cohort investigated10.Exome sequencing (ES) has revolutionized the diagnosis of genetic disorders, and has contributed to the discovery of new Mendelian-disease genes11- has been implemented in many clinical genetics laboratories, becoming a robust and cost-efficient diagnostic tool12, despite its inherent challenges and limitations13.The clinical exome sequencing -which comprises genes associated to known clinical phenotypes and covers > 98% of variants identified on targeted next-generation sequencing (NGS) panels14. The Genetics Department of the Fundación Jimenez Diaz University Hospital (FJD-HU) in Madrid (Spain) is a well-established and experienced genetics laboratory that integrates cytogenetic and molecular techniques. Before the NGS-era, the analysis of point variants and the sequencing of genes were offered by Sanger sequencing. Gradually, after the NGS implementation, its protocols have been adapted to this new technology. Nowadays, we offer all our patients an initial clinical evaluation and a molecular testing for each disorder being the NGS and, specifically CES, the first diagnostic approach for many of them.A recent overview of the different genetic approaches used to evaluate undiagnosed diseases has been published, emphasizing on how NGS can be incorporated in routine clinical practiceHere, we present a 5-year retrospective review of 4974 clinical exomes performed (from October 2016 to April 2021), analysed and informed in our laboratory. We describe our NGS cases, their distribution, and the global and the specific diagnostic rate (DR) achieved for each type of disorder. We also remark the importance of having an experienced team of clinical and molecular geneticists and emphasise the importance of appropriate communication with fellow clinicians, in an increasingly complex and demanding field.All patients were referred to the Genetics Department of the Fundación Jimenez Diaz University Hospital  with a clinical suspicion of a genetic disorder from different Clinical Departments. All underwent full clinical genetic evaluations. Only if indicated and after an adequate pre-test genetic counselling, patients were molecularly studied. Written informed consent was obtained from each participant in accordance with institutional requirements. The study was reviewed and approved by the Research Ethics Committee of HU-FJD and fulfilled the principles of the Declaration of Helsinki.Whole peripheral blood samples were collected from probands (n = 4974). Data on sex, age and available clinical indication for genetic testing was retrospectively recollected.Illumina In, San Diego, CA, USA) (N = 612), and the Clinical Exome Solution  (N = 4362) that capture 4813 and 4900 genes related to inherited diseases, respectively. Over 95% of the captured genes overlapped in both approaches. The detection of Copy Number Variants (CNVs) was only possible in samples sequenced with CES.Genomic DNA was extracted following standard procedures. From October 2016 to April 2021, clinical exomes were performed using the TruSight One Sequencing panel  and small insertions/deletions (indels) in TSO samples was performed using the Burrows-Wheeler alignment (BWA) Enrichment App, which provides industry-standard alignment. Variant calling was based on the Genome Analysis Toolkit (GATK). An in-house Sophia Genetics pipeline was used for the analysis of single nucleotide variants in CES samples. The detection of CNVs in CES samples was also performed using an in-house Sophia Genetics method based on a hidden Markov model. Specifically, the method evaluates the coverage levels of the target regions across all samples within the same sequencing run. The coverage is normalized by sample and target region, thus enabling CNV calling. A minimum of 8 samples per run is required.15. In the remaining samples, only indels and bad-quality NGS variants  were further confirmed.Informed NGS variants in the first 825 clinical exomes of fully characterized cases were validated by Sanger sequencing before reportingFor CES samples, all CNVs were confirmed before being reported, either by Multiplex ligation-dependent Probe amplification (MLPA) or by array-CGH (60 k), depending on the availability of the MLPA kit and the size of the CNVs.The analysis of the mitochondrial DNA or the clinical exome analysis in trio was not performed in any of the analysed patients.Illumina-) and the SOPHiA DDM™  platforms were used for the analysis of variants.The Variant Studio (-16: benign (B), likely benign (LB), unknown clinical significance (VUS), likely pathogenic (LP) and pathogenic (P). Only P and LP variants, as well as VUS clearly related with the phenotype were reported.Variant interpretation was performed by our team of experienced molecular geneticists, making use of population and clinical databases , the literature and other tools such as Varsome or InterVar. Variants were classified according to the ACMG criteria in the 5 established categoriesIn some cases, when only a LP or P variant was detected in a recessive related-gene, additional testing such as MLPA, array CGH or Sanger sequencing of uncovered regions was performed to attempt detecting the second allele, specifically in samples analysed by TSO as CNVs are not detected with this technology.Clinical virtual panels were developed based on public database  and medical specific literature. Specifically, retinal dystrophy panels were based on the RetNet database. Each panel was periodically re-evaluated—at least once a year- and thus, the panel content changed over time during the study.“Molecular diagnosis” was defined as the presence of a P or LP variant in a dominant or X linked gene; as two P or LP, one P and one LP variant in a recessive gene, respectively, sex regardless. This group was used to define the global DR in our cohort and among the different groups of diseases.“Potential molecular diagnosis” was stablished with a VUS in a dominant gene potentially explaining the phenotype, two VUS in a recessive gene potentially explaining the phenotype or VUS in a X-linked gene.“Partial molecular diagnosis” includes cases with only one P or LP variant in a recessive gene  potentially explaining the phenotype.“No molecular diagnosis” was defined when no variants were reported or when either a monoallelic P, LP or VUS in recessive gene or a VUS in a dominant gene not clearly related with the clinical suspicion was detected.For data analysis, we established different groups of diseases based on the more demanding Clinical Departments  and/or on the more demanding diseases . Within the majority of them, different subgroups of diseases were established.A summary an overview of the distribution of the 4974 analysed cases are shown in Fig. A total of 4974 patients  were analysed. The mean age (± SD) at diagnosis was 36.1 ± 22.8 years, ranging from 0 to 96 years of age.Since the implementation of the NGS in the laboratory, we have faced a continuous and exponential growth in the number of clinical exomes requested. In the last 3 years, we approximately performed 1300–1400 clinical exomes per year Fig. .Figure 2We analysed a total of 4974 clinical exome panels. The two more frequent groups of diseases were Ophthalmological  and Neurological disorders , followed by Cardiological cases including those with clinical suspicion of Marfan syndrome, Noonan or other Rasopathies . A total of 272 (5.47%) Endocrinological disorders, and 150 (3.02%) Kidney disorder cases were analysed. Skeletal dysplasias (n = 134), Hearing impairment (n = 133), Cystic Fibrosis (n = 80) and Familial Hypercholesterolemia (N = 70) corresponded to 2.69%, 2.67%, 1.61% and 1.41% of total cases, respectively. The remaining 346 cases (6.96%) corresponded to a miscellany of disorders that includes Autoinflammatory, Metabolic and Dermatology cases among others  and 9.25% (n = 460/4974) of total cases, respectively.In 280 cases , a partial molecular diagnosis was achieved. No molecular diagnosis was established in 57.16% of samples (2843/4974), including few cases with a VUS in a recessive reported gene with doubtful association with the clinical suspicion , a deletion or duplication was detected in 29 cases (1.8%), of which 23 (12 deletions and 11 duplications) belonged to the molecular diagnosis group . Interestingly, the chromosomes with more detected CNVs were chromosome 15 and X (8 vs 4 cases respectively), followed by chromosomes 1 and 17 (3 cases each).We observed that the DR was highly dependent on the indication of study and on the knowledge of the disease. The best molecular DR was achieved in cases derived from Ophthalmology  followed by familial hypercholesterolemia cases  and hearing impairment . In Nephrology (n = 38/150) and Neurological cases (n = 254/1529) we achieved a DR of 25.33% and 16.61%, respectively  correspond to cases with a VUS in a dominant gen, we could estimate that 17% of potential molecular cases will be reclassified to molecular cases, giving a global DR of around 29% (1469/4974), which falls into the rates previously reported by other groups.ES is a robust and cost-efficient diagnostic tool but, based on large cohort studies, it only provides an overall molecular diagnosis in 25–30% of cases7. Based on the literature, it is estimated to be approximately of 36% in developmental disorders20 but around 50% and 58% in hearing loss and retinal dystrophies, respectively22. In our cohort, we had the higher DR in ophthalmological and familiar hypercholesterolemia cases (43.12% and 38.57% respectively) compared to other disorders such as neurological cases (16.61%), cardiology (17.13%) or others (20.81%) . Probably, we missed others due to the limitations of the CNVs detection by ES, which depends on the capture regions, the homogeneity and the coverage obtained for each sample.CNVs have been associated with a broad range of pathological conditions, accounting approximately for 13% of the human genome alterations24. The pipeline used during the analysis is also essential to improve diagnosis, as reflected in our capacity to increase the DR of previously unsolved cases in 2.5%, 3.2% and 4.4% of cancer, cardiovascular and retinal dystrophy cases respectively, as recently reported by our group25. In addition, reanalysis of ES data represents a powerful approach to identify additional diagnosis and improve patient care28. According to recent studies, exome reanalysis improves the DR in approximately 12%, with reported increases ranging from 5 to 26%29. It is known that this increased DR is mainly due to the discovery of new disease-genes, the use of better bioinformatics tools, such as large databases and improved pipelines that help in the reclassification of variants, as previously demonstrated by our group31, and the availability of additional phenotype data, among others16. In this line, prioritized features and genes using Human Phenotype Ontology terminology have been proposed as a helping diagnostic tool33. In addition, good communication with the clinicians ordering tests and an continuous genomic data sharing, is essential for the discovery of new genes -as already demonstrated with the GeneMatcher tool36-and for a better interpretation of variants that will definitely improve our diagnosis process3. Given the evolving nature of the genomic field, it is also essential to implement the exome reanalysis as part of routine clinical practice37.The NGS diagnosis success strongly relies on the analysis and the strategy used for gene selection and variant filtering14. Although ES is gradually being optimized, it has limitations and in some cases generates laboratory’s errors difficult to avoid38. In this situation, whole genome sequencing (WGS) is becoming an attractive alternative as it is presumably able to detect more potential pathogenic variants than ES41. Thanks to its greater breadth of coverage, genome sequencing (GS) is better than ES to detect single nucleotide variants and CNVs, also allowing the detection of variants in non-coding regions and chromosome rearrangements, all of which have been demonstrated to be involved in human disease43. It is not casual that if we exclude cystic fibrosis cases due to its high prevalence in the general population, the higher partial molecular diagnosis is achieved in ophthalmological (9.2%), kidney (7.33%) and hearing impairment (7.52%) cases, in which the highest DR is reported in the literature. Undoubtedly, the analyses of non-coding regions or improved CNVs would help us to detect the second missed allele, increasing our global DR with the corresponding benefit for the patients.Big efforts are still needed to improve our genetic DRHere, we present the DR of the CES we routinely perform in our clinical practice. Probably, the case selection, the continuous communication with clinicians and the expertise of our genetics team, make the difference in favour of our global estimated DR of 29%. Nevertheless, this overall DR is still low and direct efforts on the analysis and interpretation of NGS are required. Large databases, improved pipelines, data sharing and further investigations for a deeper understanding of ES, GS and other focused genetics techniques are needed in order to improve our global DR in the benefit of patients.


This study evaluated the effects of dietary butyrate supplementation and oral nonsteroidal antiinflammatory drug (NSAID) administration on uterine inflammation and the interval from calving to first ovulation . We hypothesized that a combination of dietary butyrate and oral NSAID would reduce uterine inflammation and decrease ICFO. Sixty-five cows were enrolled in a 2 × 2 factorial design and assigned to receive an iso-energetic diet containing a supplement of either butyrate  or control  at 1.42% of diet dry matter, during the calving transition period from −28 (±3) to +24 (±3) days in milk . At 12 to 24 h postcalving, cows received an oral NSAID (1 mg of meloxicam/kg of BW) or a placebo (food dye). Ovarian ultrasonography was performed weekly from 14 DIM until first ovulation or up to 56 DIM. Endometrial cytology was performed at 28 DIM to assess uterine inflammation based on polymorphonuclear leukocytes (PMN). No interactions were detected between treatments. The proportions of cows with high (>18%) endometrial PMN did not differ between butyrate and control diets or between NSAID and placebo. Mean (± standard error of mean) ICFO did not differ between butyrate (28 ± 2 d) and control (25 ± 2 d) or between NSAID (29 ± 2 d) and placebo (24 ± 2 d). However, the ovulation rate up to 56 DIM  established by survival analysis tended to be lower in NSAID than in placebo. In conclusion, dietary butyrate supplementation and oral NSAID administration did not reduce endometrial inflammation or the mean ICFO, but NSAID-treated cows tended to have a lower rate of ovulation up to 56 DIM. Postpartum dairy cattle regularly experience microbial invasion of the uterus, characterized by increased PMN in the uterine lumen . AlthougShort-chain fatty acids, such as butyrate, activate and regulate the immune system , and cowNSAID), such as acetylsalicylate, carprofen, and meloxicam, has been explored to manage endometritis, and NSAID have been reported to reduce inflammation .Parenteral administration of nonsteroidal antiinflammatory drugs  in 2019–2020. All procedures were approved by the University of Alberta Animal Care and Use Committee for Livestock (AUP00003364). This experiment was conducted in tandem with Sixty-five  Holstein cows were blocked by parity and expected calving date and randomly assigned to 1 of 2 iso-energetic diets containing fatty acid-coated calcium butyrate  or a control supplement [1.04% commercial fat  and 0.38% calcium carbonate] at 1.42% of diet DM. Prepartum and postpartum diets, and details describing diet formulation and dietary butyrate concentration determination, are included in Cows received an oral NSAID  administered at 1 mL/15 kg of BW in carrier solution, equivalent to 1 mg of meloxicam/kg of BW) or a placebo (food dye at 1 mL/15 kg of BW in carrier solution) at 12 to 24 h after calving using an oral drench gun. Study personnel were blinded to NSAID treatment until completion of data analysis. Treatment groups were (1) butyrate + NSAID (n = 15), (2) butyrate + placebo (n = 18), (3) control + NSAID (n = 14), and (4) control + placebo (n = 18).Ovarian structures were evaluated once weekly by transrectal ultrasonography from 14 ± 3 d postpartum (DPP) until the first ovulation was confirmed, or until a maximum of 56 ± 3 d. Locations of major ovarian structures  were recorded, and ovulation was confirmed by the appearance of a corpus luteum in the ovary. Vaginal mucus samples were collected using a Metricheck device (Simcro Tech Ltd.) at 14 and 28 d, and its appearance and odor were scored as per Samples for endometrial cytology were obtained at 28 d using a cytobrush  modified for use in large animals as described by P ≤ 0.05 and tendencies were declared when P > 0.05 but ≤0.10.Statistical analyses were conducted using SAS . Normality of data was determined using the UNIVARIATE procedure. Binomial and continuous dependent variables were modeled against the fixed effects of independent variables  and analyzed using GLIMMIX procedure of SAS. For binomial dependent outcomes, the model was specified as binomial (dist = binary link logit), and the ilink option with Tukey's adjustment used to obtain corresponding least squares means by parity, dietary, and drug treatment groups. As none of the interactions was significant, the fixed effects of interactions were removed from the final model. The differences in the proportions of cows by vaginal mucus characteristics at 14 and 28 d were also analyzed by GLIMMIX procedure with Tukey's adjustment to obtain LSM by parity, dietary, and drug treatment groups. In addition to determining the proportion of cows that ovulated at 14, 21, 28, 35, 42, 49, and 56 DPP using the aforesaid GLIMMIX procedure, the probability of ovulation up to 56 d DPP was evaluated by the Kaplan-Meier survival analysis (LIFETEST procedure) and tested by a Cox proportional-hazards model (PHREG procedure). During data collection, 7 cows  were treated with antibiotics or NSAID  for health disorders. Data for these cows were included up to when the above interventions occurred but removed from any statistical analysis after treatment. Significance was declared at P = 0.90) or drug treatment . The proportions of cows distributed by vaginal mucus characteristics on d 14 and 28 also did not differ  endometrial PMN did not differ by dietary  or between NSAID and placebo . The ovulation rate up to 56 DPP by survival analysis did not differ between butyrate and control . However, the ovulation rate up to 56 DPP tended to be lower in NSAID than in placebo . There were no differences in the interval from calving to the first detection of ovarian follicles of 10-mm diameter in butyrate compared with control  or in NSAID compared with placebo . Similarly, the interval from calving to the first detection of 16-mm diameter follicles did not differ in butyrate compared with control  or in NSAID compared with placebo . The absence of treatment differences in the intervals from calving to detection of 10- and 16-mm follicles indicates that the tendency for a lower ovulation rate up to 56 DIM in NSAID was not because of impeded recruitment of preovulatory size follicles.The cumulative proportions of cows that ovulated at each weekly interval  and the Butyrate has been proposed to improve reproduction in cattle as it is an energy source ; howeverLimited data are available on the effects of NSAID on uterine inflammation in dairy cattle; however, those who have assessed it found no changes , similarIn the present study, NSAID was administered orally, whereas in much of the existing research NSAID was given through s.c. or i.m. routes. Although oral meloxicam has a similar therapeutic onset to the injectable form, the oral formulation has a longer half-life  and a significantly longer lasting action . InjectaWe acknowledge that the differences in the modes of action of different NSAID formulations may have also contributed to the differences among studies. The present study used meloxicam, whereas other studies have evaluated carprofen  and sodiCows given the NSAID tended to have a reduced ovulation rate up to 56 DIM compared with cows given placebo in the present study. It has been reported that NSAID use delays ovulation in rodents  and humaIt has been suggested that NSAID are likely more effective for cows experiencing calving difficulties  or inflaIn conclusion, neither dietary butyrate supplementation nor oral NSAID administration reduced endometrial inflammation or reduced the mean ICFO. However, NSAID-treated cows tended to have a lower ovulation rate up to 56 DPP than cows given placebo. Considering the lack of power in the present study, further research with a larger sample size is warranted to understand the effects of NSAID on uterine inflammation and ovarian function.