Correlations between fluctuations in resting state BOLD fMRI signals are interpreted as measures of functional connectivity (FC), but the neural basis of their origins and their relationships to specific features of underlying electrophysiologic activity, have not been fully established. In particular, the dependence of FC metrics on different frequency bands of local field potentials (LFPs), and the relationship of dynamic changes in BOLD FC to underlying temporal variations of LFP correlations, are not known. We compared the spatial profiles of resting state coherences of different frequency bands of LFP signals, with high resolution resting state BOLD FC measurements. We also compared the probability distributions of temporal variations of connectivity in both modalities using a Markov chain model-based approach. We analyzed data obtained from the primary somatosensory (S1) cortex of monkeys. We found that in areas 3b and 1 of S1 cortex, low frequency LFP signal fluctuations were the main contributions to resting state LFP coherence. Additionally, the dynamic changes of BOLD FC behaved most similarly to the LFP low frequency signal coherence. These results indicate that, within the S1 cortex meso-scale circuit studied, resting state FC measures from BOLD fMRI mainly reflect contributions from low frequency LFP signals and their dynamic changes. In addition, the identification of patterns of correlated, low frequency fluctuations in BOLD signals in a resting state has provided a remarkable tool to assess functional connectivity between regions in the brain, providing insights into how distributed cortical areas work together to achieve specific functions7. However, BOLD signals are produced by hemodynamic effects that are only indirect indicators of underlying neural activity. Previous studies of the relationships between simultaneously recorded electrophysiological activity and hemodynamic changes in sensory systems have suggested that BOLD signals temporally correlate strongly with local field potentials11. Furthermore, our recent studies have indicated that at a columnar level, BOLD signals recorded at high magnetic field faithfully reflect underlying neuronal activity during stimulation, and the spatial profiles of local correlations in BOLD signals match those of LFP signals from the same region12.Functional MRI based on detecting BOLD (blood oxygenation level-dependent) signal changes is a well-established neuroimaging technique for detecting and spatially characterizing brain regions that change their levels of activity in response to specific external tasks13. While our own and other studies have revealed how resting state fMRI connectivity spatially corresponds to correlations between LFPs, there have been few previous reports of what specific characteristics of LFPs best match BOLD correlations, or what dynamic features in the frequency contents of LFPs best align with temporal changes in BOLD metrics. Previous non-human primate brain studies have found that different frequency bands of LFP activities are linked to different neural information processes14. Typically, prominent LFP oscillations span a broad range of frequencies that by convention is separated into specific delta (1–4 Hz), theta (5–8 Hz), alpha (9–14 Hz), beta (15–30 Hz), gamma low (30–50 Hz), gamma high (50–100 Hz), and gamma very high (100–150 Hz) bands. In stimulation or task conditions, there have been consistent reports of close relationships between increases in BOLD signal amplitudes and increases in power of gamma band LFP (30–150 Hz) of LFP signals16. In a resting state, however, the relationships between resting state BOLD and spontaneous LFP activity remain elusive. While some studies suggest that lower frequency band fluctuations (<20 Hz) may predominantly correspond to changes in BOLD signals18, others suggest that slow changes in the gamma band of LFP signals contribute most to the spontaneous fluctuations in BOLD signals20. Moreover, resting state correlations of BOLD signals between regions appear to vary over time, and it has been postulated that these apparent dynamic changes of fMRI connectivity represent another level of neural modulation25. To date, few studies have directly compared the dynamic variations of connectivity seen in fMRI with corresponding properties of LFPs, nor have they established which LFP frequency bands are most relevant to these BOLD temporal dynamic changes27. No study, to our knowledge, has compared directly both the spatial relationships and the dynamic features of functional connectivity between BOLD signals and different frequency bands of LFPs within a well-defined, mesoscale functional network in a resting state.The interpretation and application of fMRI-based functional connectivity studies rely on our understanding of the precise relationships between synchrony of local neural activity and correlated fluctuations in BOLD signals. To date, few studies have directly investigated the local spatial and temporal dynamic relationships between resting state BOLD signals and different frequency bands of local field potentials12. Here we examine those relationships more closely and address two specific new questions. First, to what extent do temporal dynamic changes in functional connectivity in BOLD and LFPs show similar quantitative characteristics, which is important to interpret the significance of dynamic changes in resting state correlations. Second, what specific frequency bands of LFP coherences most parallel the dynamic features of BOLD functional connectivity in a resting state, which is currently unclear given previous studies implicating different origins and functional relevance of different LFP frequencies28.We previously reported comparisons of local spatial profiles at columnar level between stimulus-evoked amplitudes and resting state correlations for both BOLD signals and broadband LFPsSpecifically, we acquired high resolution (sub-millimeter) BOLD images at high field (9.4T) and multielectrode array LFP recordings from the areas 3b and 1 of primary somatosensory cortex (S1) in non-human primates. We measured the spatial extents of BOLD activations in response to tactile stimuli as well as the spatial profiles of single-voxel local correlations in a resting state. We then quantitatively compared these with the spatial extents of LFPs in each of seven frequency bands, and their inter-electrode coherences, in the same conditions and brain regions. We also compared dynamic features of the resting state connectivity measures in both modalities by using a modified Markov chain model-based approach combined with a sliding window correlation analysis. We aimed to quantitatively compare the time-varying patterns of functional correlations between BOLD and frequency-specific LFPs in individual monkeys, and to determine what frequency ranges of the LFP coherence dominate changes in dynamic BOLD functional correlations in two S1 cortices.p < 0.05; Fig. p < 0.05; Fig. Figure 25, but we also evaluated whether smaller window sizes would reveal additional apparent changes in resting state connectivity. The dynamic patterns of functional correlations were very similar. The low frequency LFP coherence changed more frequently than for high frequency components. We quantified this difference by analyzing the Fourier spectral composition of the coherence between areas 3b and 1 in resting state BOLD signals and frequency-specific LFPs. We compared the cumulative percentage of frequency components derived from resting state BOLD correlations with frequency-specific LFP coherences. Figure Figure p < 0.01). These results indicate that within S1 cortex, the dynamic changes of BOLD functional correlations behaved more closely to those of low frequency than high frequency LFP coherences. We also applied the band-limited power . In addition, evidence has shown that the neural oscillations of frequency-specific LFP signals may be generated by various types of neurons in different pathways37. The primary somatosensory cortex is a cortical region receiving a convergence of anatomical connections from structures in the cortico-thalamic and cortico-cortical loops40. Low frequency oscillations can occur in a cortico-thalamic feedback loop. These slow oscillations are thought to be modulated by global neuronal inputs41. However, high frequency LFPs, such as gamma-waves, most likely arise locally within cortical microcircuits that include pyramidal cells and interneurons44, and therefore, present a more constrained local spatial correlation profile. Our results also showed that in a resting state, the local correlation profiles of broadband LFPs were not significantly different from those of low frequency LFPs (1–15 Hz) and gamma low band (30–50 Hz). Furthermore, we directly compared the spatial profiles between stimulation and resting state conditions in both areas 3b and 1, respectively. We found that in area 3b, the spatial extents of resting state electrode-electrode coherence in the broad band (1–150 Hz) and the frequency ranges between 5 and 30 Hz were significantly larger than those of stimulation responses. However, in the frequency ranges between 50 and 150 Hz, the spatial extents of resting state coherence were significantly smaller than those of stimulation responses, a feature that was not statistically significant in area 1. These comparisons show that in S1 cortex, low frequency LFP signal fluctuations spatially dominate resting state LFP coherence, while stimulus responses are dominated by high frequency LFP responses. These relationships could potentially vary in different brain regions. For example, in the hippocampus, strong theta oscillations (5–8 Hz) are the most prominent feature in LFP signals and are believed to be critical for hippocampal-cortical interactions46, while in visual cortex, fast oscillations in the gamma band between 30 and 90 Hz are dominant during resting states47.The local coherence profile of LFP signals within S1 cortex in a resting state directly measures the contributions from the intrinsic spatial distribution of the neural activity involved, and potentially reflect how neurons spatially work together at a columnar level. No previous study, to our knowledge, has reported the spatial relationships of LFP signals in different frequency ranges during stimulation or in a resting state. We found that in a resting state, the local correlation profiles of LFP signals from the frequency ranges between 50 and 150 Hz were significantly smaller than those of the frequency ranges between 5 and 30 Hz. This suggests that in a resting state, neurons generating high frequency LFP signals (50–150 Hz) may intrinsically communicate more focally within a finer-scale cortical microcircuit49, and some electrophysiological studies have shown that delta (1–4 Hz) and alpha (9–14 Hz) generators are strongly represented in superficial layers51. It will be valuable to investigate the laminar profile of LFP signals in different frequency ranges, and the spatial and temporal relationships between cortical layer-specific BOLD signals and their corresponding LFPs. Lastly, it is worth noting that in animal studies, in order to obtain reliable interpretations of functional connectivity changes, it is necessary to estimate the effects of anesthetics on the resting state fMRI signals. In our lab’s previous publication52, the authors observed universal dose-dependent suppressive effects of isoflurane anesthesia on cortical regions. Although it is difficult to form a standard anesthesia range across studies, for the purposes of this study, we maintained an appropriate isoflurane level (0.7–0.8%) for imaging of the S1 cortex as stable as possible during all functional scans.We identified seed voxels and electrodes for calculating resting state correlations in BOLD and LFP measurements separately, according to the stimulus-evoked activation maps in each monkey. In Figs Saimiri bolivians, SM-ZT, SM-TE, SM-FZ) were included in this study and each underwent functional MR imaging, micro-electrode electrophysiological mapping and multi-channel microelectrode array recording sessions. Animals were pre-anesthetized with ketamine hydrochloride (10 mg/kg)/atropine (0.05 mg/kg) and then anesthetized with 0.5–1.5% of isoflurane delivered in a 30:70 O2:N2O mixture, to maintain a stable physiological condition for both MRI scans and electrophysiological acquisitions. Although the actual level of isoflurane may vary during the scan and across animals, we typically maintained a light level of anesthesia at ≈ 0.7–0.8% isoflurane during all functional acquisitions and electrophysiological signal recordings. The anesthetized animals were intubated and artificially ventilated. After intubation, each animal was placed in a custom-designed MR cradle with its head secured using ear and head bars. Lactated Ringer’s solution was infused intravenously (2–3 ml/h/kg) to prevent dehydration during the course of the study. Blood oxygen saturation and heart rate , electrocardiogram, end-tidal CO2 , and respiration  were monitored and maintained at stable levels. Body temperature was monitored (SA Instruments) and maintained between 37.5 and 38.5 °C via a combination of a circulating water blanket . Animals were carefully monitored from the time of induction of anesthesia until full recovery. Analgesic (buprenorphine) is for 3 days post-surgery. Detailed procedures have been described in our previous publication55. All experimental procedures were in compliance with and approved by the Institutional Animal Care and Use Committee of Vanderbilt University, and followed the guidelines of the National Institute of Health Guide for the care and use of laboratory animals.Three squirrel monkeys , leaving the glabrous surfaces available for vibrotactile stimulation by a blunt plastic probe (2 mm diameter) connected to a piezoelectric vibrator . The vibrators were driven by Grass S48 square wave stimulators  at a rate of 8 Hz. For fMRI data acquisitions, vertical indentations (0.34 mm displacement) of a probe at 8 Hz rate (with 20-ms pulse duration) were presented as blocks of 30 sec on and then 30 sec off. Seven blocks were typically presented within one imaging run. Multiple runs were typically acquired. The timing of the presentation of stimuli was externally controlled by the MR scanner and was synchronized to image acquisition. The same stimulus presentation paradigm was applied during later electrophysiological recordings. Each stimulation trial consisted of 20 repetitions of 30 sec on/off blocks. Typically, 10 trials were collected for each digit. Detailed protocol has been described in our previous publication2*-weighted gradient-echo structural images  were acquired to identify venous structures on the cortical surface to help locate S1 cortex and as structural features for co-registration of fMRI images  and exported to Matlab (Mathworks) for analysis12.MR imaging was performed with a 9.4 T 21 cm bore magnet and Varian/Agilent Inova spectrometer , using a 3-cm surface transmit-receive coil secured over S1 cortex. Scout images obtained using a fast gradient-echo sequence were used to define a volume covering S1 cortex  in which static magnetic field homogeneity was optimized, and to plan four oblique slices for structural and functional imaging. T56. No spatial smoothing was applied. Resting state BOLD signal time series were further filtered with a low-pass filter (0.01–0.1 Hz). The top image slice, in which areas 3b and 1 reside, was analyzed for activations. For stimulation runs, we computed activation maps based on the percentage of BOLD signal change between pre-stimuli periods (seven out of ten imaging volumes before stimulus onset) and stimuli presentation periods (10 image volumes). The activation response map  for further sub-voxel processing and display. Within each fMRI stimulation run, the percentage of BOLD signal changes in each of the cortical areas (areas 3b and 1) was normalized to its own maximum value. Normalized percentage BOLD signal changes were then used in the group analyses. Detailed fMRI data acquisition and preprocessing procedures have been described in our previous publication12.Standard preprocessing steps were applied to the raw fMRI signals. Slice timing correction was performed after slice-by-slice motion correction. Six translation and rotation parameters were used to regress out temporal variations caused by motion. Time courses were drift corrected using a linear model fitted to each time course. The RETROICOR method was used to correct for physiological noise, using the respiration and cardiac patterns recorded during the scanmap Fig.  was then10) and used for further analysis. To estimate more accurately the mean power of the steady-state LFP responses, we excluded the data from the first 10 sec after the stimulus onset in our calculation, according to previous observations57. For LFP resting state coherence analysis, magnitude squared coherence in each frequency band was calculated between each selected seed electrode and all other electrodes . As seen in Fig. des Fig. . Detaile12. To extend our previous analyses, the same fitting methods were used to quantify the spatial extents of LFP signals in each of the eight frequency bands. We identified activation foci in areas 3b and 1, whose shapes appear to be elliptical. In each animal, activation maps of three different digit tips  were typically obtained. A threshold of 0.7 was used to identify the center of mass in each activation focus. The axis along the individual digit activation centers was then determined as the major  axis within each area (3b and 1). The perpendicular anterior-posterior direction of this axis was then determined as the minor axis. From the fitting, the major  and minor (in the anterior to posterior direction) axes of the ellipse were determined, and all values above 0.2 of normalized percentage signal change along the major and minor axes within the sub-region were used in the subsequent Gaussian fitting. The point spread functions  of the single digit tactile stimulation evoked BOLD fMRI activation maps were measured in both areas 3b and 1. The coordinates of the center of mass, and the major and minor axes of the ellipse were determined by fitting. The spatial distributions of percentage of BOLD signal changes along the major and minor axes were then fit with Gaussian functions. The FWHM of each fitted Gaussian was then computed. For resting state fMRI runs, ROI seeds were identified based on the vibrotactile stimulus-evoked activation maps. The voxels with the highest % BOLD signal change were chosen as the seeds for each digit in either areas 3b or 1  for further sub-voxel processing. Within each LFP stimulus recording run, the percentages of signal power changes of cortical activation areas were normalized to their maximum values during each run. For resting-state LFPs, seed electrodes were identified based on the array stimulus response maps as those showing the largest percentage signal power changes during single digit stimulations in both area 3b and area 1. Functional connectivity maps based on functional coherence between each seed and the rest of the electrodes were then computed. We then used the same fitting as described above to describe the spatial profiles of resting-state LFP coherences of this area.To quantify the dimensions of LFP activities in each frequency band, the same ellipsoid Gaussian PSF (FWHM) fitting was performed58.Previous studies have shown that resting state correlations in BOLD signals between regions may vary over time. Here we compared whether LFPs show similar dynamic changes, and examined which (if any) specific LFP frequencies most closely match those of fMRI signals. To do this, we consider the instantaneous degree of correlation between regions to represent a brain state, so that changes in connectivity correspond to transitions between brain states. The number of such states is unknown but we assume they may span the range between perfectly correlated and perfectly anti-correlated, and this range can then be divided into a finite number of discrete intervals. Measurements of the variations over time in correlation values allow the construction of a probability distribution of the states, along with probabilities for transitions from any one state to another. A discrete-time Markov chain model is a tool for representing probability distributions over a sequence of states in which the probability of moving to the next state depends only on the present and not on the previous states25. The sampling variability and statistical efficiency of the sliding window technique has been recently investigated59. In the current study, resting state correlations between the BOLD time series derived from areas 3b and 1 within S1 cortex were calculated using a sliding window with a window size of 60 sec. The selection of the window size was based on simulations and a two sample Kolmogorov-Smirnov (K-S) statistic in our previous studies25 which established this was sufficient to attenuate spurious variations while maintaining low frequency features of interest. The window was shifted in time in 3 sec increments  along the entire time series and the correlation coefficient was recalculated. As the sampling distribution of Pearson’s r is not normally distributed, correlation coefficients were converted to z-scores using Fisher’s z transform25. The z-scores from −1 to 1 were binned into 50 equal steps, which represent 50 putative brain states characterized by their differing degree of correlations between areas 3b and 1. The selection of 50 was arbitrary but shown to not be a critical parameter. A similar sliding window technique was used to classify the LFP signals. In this analysis, however, the magnitude squared coherence values for each frequency band were computed from the raw LFPs29 instead of Pearson’s correlation coefficients. We then quantified the resemblance of the temporal variations in the correlations between BOLD and frequency-specific LFP signals by constructing the empirical cumulative distribution functions (ECDFs) for jumps between states, which represent the probability functions for single-step transitions between different degrees of connectivity. A two-sample Kolmogorov-Smirnov (K-S) statistic60 was used to evaluate whether the BOLD and LFP ECDFs were derived from the same population with a specific distribution, based on measurements of the distance between two ECDFs. This test is sensitive to differences in both the location and shape of the ECDFs of the two samples60. Lower K-S values between BOLD and LFP in a certain frequency band suggest increased similarity in the nature of the dynamic changes in functional connectivity.Figure


Endogenous signaling molecules derived from lipids, peptides, and DNA, are important regulators of physiological processes during pregnancy. The effect of their collective impact on preterm birth (delivery < 37 weeks gestation) is understudied. We aimed to characterize the associations and predictive capacity of an extensive panel of eicosanoids, immune biomarkers, oxidative stress markers, and growth factors towards preterm birth and its subtypes. We conducted a cross-sectional study of pregnant women (recruited < 15 weeks gestation) in the LIFECODES birth cohort, which included 58 cases of preterm birth and 115 controls that delivered term. Among the cases there were 31 cases who had a spontaneous preterm birth (cases who had spontaneous preterm labor and/or preterm premature rupture of membranes) and 25 that had preterm birth associated with aberrant placentation (cases who had preeclampsia and/or intrauterine growth restriction) and 2 cases that could not be sufficiently categorized as either. We analyzed single biomarker associations with each preterm birth outcome using multiple logistic regression. Adaptive elastic-net was implemented to perform a penalized multiple logistic regression on all biomarkers simultaneously to identify the most predictive biomarkers. We then organized biomarkers into biological groups and by enzymatic pathways and applied adaptive elastic-net and random forest to evaluate the accuracy of each group for predicting preterm birth cases. The majority of associations we observed were for spontaneous preterm birth, and adaptive elastic-net identified 5-oxoeicosatetraenoic acid, resolvin D1, 5,6-epoxy-eicsatrienoic acid, and 15-deoxy-12,14-prostaglandin J2 as most predictive. Overall, lipid biomarkers performed the best at separating cases from controls compared to other biomarker categories . Among the enzymatic pathways that differentiate eicosanoid metabolites, we observed the highest prediction of overall preterm birth by lipoxygenase metabolites using random forest , followed by cytochrome p450 metabolites using adaptive elastic-net . In this study we translate biological hypothesis into the language of modern machine learning. Many lipid biomarkers were highly associated with overall and spontaneous preterm birth. Among eicosanoids, lipoxygenase and cytochrome p450 products performed best in identifying overall and spontaneous preterm birth. The combination of lipid biomarkers may have good utility in clinical settings to predict preterm birth. Babies who are born preterm have increased risk for health conditions later in life, including neurodevelopmental and sensory disorders, physical disabilities, cardiovascular disease, and impaired respiratory function4. Live births are broadly classified as preterm if they occurred at less than 37 weeks gestation, and there is great heterogeneity in clinical presentation and other circumstances contributing to preterm birth. Therefore, differentiation of preterm birth cases by clinical subtypes provides greater insight on the underlying antecedent mechanisms of preterm delivery. For example, spontaneous preterm births can involve spontaneous preterm labor or premature rupture of membranes, which is determined by the presence of vaginal pooling and either documentation of nitrazine-positive testing or ferning followed by regular uterine activity5. Whereas preterm birth associated with aberrant placentation is dominated by the presentation of preeclampsia (encompassing onset of hypertension and proteinuria) or intrauterine growth restriction5.Preterm birth is a significant public health issue that occurs in approximately 10% of live births in the U.S., and remains the leading cause of neonatal mortality6. Furthermore, several biomarkers of oxidative stress and inflammation serve as signaling molecules that are integral for not only regulating maternal hormones but also facilitating cross talk between the maternal circulation and fetal compartment8.During pregnancy, systemic oxidative stress – the imbalance of reactive oxygen species and antioxidant molecules – can lead to inflammation and perturbation of several physiological processes that are integral for the progression of a healthy pregnancy. For example, oxidative stress and inflammation can cause tissue damage and alter vascularization, both of which can be harmful during critical stages of pregnancy, such as placentation, quiescence, cervical ripening, and induction of labor10. For inflammation, this included the general marker C-reactive protein (CRP) as well as a panel of cytokines; for oxidative stress, this included 8-isoprostane, a marker of lipid peroxidation, and 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage. These represent only a small fraction of the repertoire of signaling molecules that regulate inflammation during pregnancy. Nonetheless, we learned that the pro-inflammatory cytokine interleukin-6 was associated with increased risk for spontaneous preterm birth, while the anti-inflammatory cytokine interleukin-10 was elevated among cases of preterm birth associated with aberrant placentation9. Among oxidative stress markers, we found that overall and spontaneous preterm birth were positively associated with 8-isoprostane and inversely associated with 8-hydroxydeoxyguanosine10.We have previously reported associations between preterm birth and select biomarkers of inflammation and oxidative stress11. Eicosanoids are intimately connected to immunological signaling molecules and inflammation, and are therefore potent regulators of physiological processes11. Animal studies also indicate overlapping consequences clustered by enzyme specific metabolites. For example, metabolites produced through LOX pathways resulted in inflammation, vascular remodeling, and altered renal function12. Furthermore, although many eicosanoids, such as prostaglandins, can cause pro-inflammatory stimulation of target tissues, some groups of eicosanoids such as resolvins can be anti-inflammatory and resolve inflammation14. Altogether, the effects of eicosanoids on inflammation and cardiovascular activity provide a mechanistic link for preterm birth15.We sought to expand our panel of biomarkers to investigate the combined effects of those previously measured biomarkers in addition to a newly measured panel of eicosanoids – a large class of lipid metabolites. Eicosanoids are metabolized from large fatty acids such as arachidonic acid and linoleic acid and produced through many enzymatic pathways, including three broad families of enzymes: lipoxygenases (LOX), cytochrome P450 (CYP450), and cyclooxygenases (COX)Our primary study aim was to evaluate a group of 65 biomarkers in association with preterm birth and its subtypes. By measuring a large panel of eicosanoids and other biomarkers of inflammation and oxidative stress, we obtain greater coverage of signaling molecules, which could potentially increase the capacity to predict preterm birth. However, there are also major challenges that arise from analyzing high dimensional data, including multicollinearity and the risk for false discoveries due to multiple testing. We integrated variable selection and machine learning methods to circumvent some of the challenges with high-dimensional data. Our secondary study aim was to focus more deeply on the biological context of observed associations by grouping our analyses based on categories of molecules, and then by enzymatic pathways within the eicosanoids panel. We hypothesized that the combination of biomarkers would predict preterm birth, and that select pathways among eicosanoids will have differential predictive capacity. We translated this hypothesis by using a grouped variable selection framework.The demographic characteristics of the subjects in this study are summarized in Table Figures Combined with the biomarkers that were associated with overall preterm birth, we observed fourteen additional lipid biomarkers associated with increased risk for spontaneous preterm birth Fig. . These bWe observed moderate to strong correlations between biomarkers Fig. . Correlaλ increased for all preterm birth , and lipid damage markers and protein damage markers exited the model last (indicating greatest importance) as the penalty on the coefficients increased Fig. . For sporth Fig. ; CYP450 rth Fig. . The groThe computed area under the Receiver Operating Characteristic (ROC) curve (AUC) is a measure of discrimination, where in our study an AUC value of 0.5 would indicate that a given combination of biomarkers has no better accuracy to predict preterm birth than chance, while an AUC value of 1 represents perfect accuracy at predicting preterm birth. While AUC values above 0.9 typically represent diagnostic tests of biomarker combinations with the greatest utility in separating cases from controls, tests with AUC values between 0.7–0.9 indicate biomarker combinations that have fair to good utility and can potentially be improved with a larger training data set or expanded measurement of additional predictive biomarkers. We reported AUC values corresponding to prediction of all preterm birth Table  and sponAccording to Table When spontaneous preterm birth was considered, the group of lipid markers again gave the largest AUC  and sensitivity  among the five major groups for each training method Table . The adaIn this study, we made two primary contributions: (1) integrated cutting edge machine learning methods and multiple inferential tools  to analyze an extensive panel of biomarkers related to inflammation, oxidative stress, and angiogenesis, in association with preterm birth and its subtypes; and (2) contextualized associations with preterm birth by grouped analyses of biomarker categories and enzymatic pathway groups for eicosanoids. To our knowledge, this is the first study to investigate eicosanoids and associated biomarkers to this degree of metabolite coverage during pregnancy in association with overall preterm birth and its subtypes. Several biomarkers were associated with overall preterm birth in single biomarker models, and adaptive elastic-net identified five biomarkers -EET, and 15DO12,14-PGJ2) to be most predictive of overall preterm birth. The overall preterm birth outcome contains multiple constituent sub-pathologies, therefore interpretation of these associations is more crude compared to focusing on preterm birth subtypes. Once we disaggregated cases by preterm birth subtypes, we learned that many of the effect estimates were largely driven by spontaneous preterm birth, where adaptive elastic-net identified four biomarkers -EET, and 15DO12,14-PGJ2) to be most predictive. Whereas for preterm birth associated with aberrant placentation, adaptive elastic-net identified two biomarkers with protective associations (PGF and LTB4). Prediction of preterm birth was most accurate with the lipid biomarkers using both adaptive elastic-net and random forest. When focusing solely on the lipid biomarkers, adaptive elastic-net showed greater accuracy in preterm birth prediction among metabolites of the CYP450 pathway, while random forest reported metabolites of the LOX pathway to be more accurate. Overall, these findings provide insight on prioritization of biomarker groups for the prediction of preterm birth, and future studies should explore CYP450 and LOX metabolites to validate these findings.17. The impact that 5-oxoETE has on target sites can vary by cell type, but broadly involve intracellular changes that lead to heightened chemotaxis – an integral process that attracts immune cells to target tissues to drive the inflammatory process18. 5(6)-EET is a fatty acid metabolite of AA produced through the CYP450 pathway and commonly originates from the liver, kidneys, and vascular endothelium19. Importantly, 5(6)-EET is a modulator of cardiovascular function and predominantly acts as an anti-inflammatory vasodilator20. We observed protective associations between 5(6)-EET and spontaneous preterm birth, which aligns with the predominant paradigm of an anti-inflammatory and anti-hypertensive profile. However, other studies have observed higher plasma and placental concentrations of 5(6)-EET or total EET in women with preeclampsia compared to normotensive women21 and non-pregnant women22. Another study found lower concentrations of 5(6)-EET in amnion of women with preeclampsia compared to laboring women that had a cesarean section23. Differences between studies underline potential nuances to 5(6)-EET activity, potentially masked by our uncertainty of concentrations at target tissues at the maternal-fetal interface.The biomarkers that adaptive elastic-net identified as most predictive for spontaneous preterm birth are all eicosanoid metabolites, with each of the three enzymatic pathways and parent compounds being represented. 5-oxoETE is a polyunsaturated fatty acid product derived from AA processing via the LOX pathway. Receptors for 5-oxoETE are highly expressed in peripheral leukocytes, lungs, kidney, liver, spleen, and placenta24. Although we observed positive associations between 15DO12,14-PGJ2 and spontaneous preterm birth, several pro-inflammatory prostaglandins  were also positively associated with spontaneous preterm birth. 15DO12,14-PGJ2 may likely be up-regulated during inflammation to help with the resolution of inflammation, thereby partly explaining why we observed positive associations with spontaneous preterm birth. A similar conclusion may be drawn from the positive association we observed between spontaneous preterm birth and the anti-inflammatory molecule RVD1, which belongs to the group of compounds called resolvins and is a LOX pathway product of DHA25. Interestingly, our findings suggest that both pro- and anti-inflammatory fatty acid metabolites are associated with increased risk for overall and spontaneous preterm birth, emphasizing the utility of evaluating these biomarkers collectively as a combination of predictors to enhance the classification of preterm birth cases early in pregnancy.The prostaglandin 15DO12,14-PGJ2 has been shown to elicit anti-inflammatory properties through inhibition of the nuclear transcription factor NFκ-B26. Previous studies observed lower levels of PGF among women with preeclampsia – one requisite for preterm birth associated with aberrant placentation – indicating that higher concentrations of PGF may potentially be protective of this type of preterm birth26. LTB4 is a leukotriene metabolized from AA through the LOX pathway, and has pro-inflammatory properties through stimulating vasoconstriction, platelet aggregation, and cellular migration of immune cells27. Although LTB4 has been previously reported to increase the risk for preeclampsia27, we observed a protective relationship between LTB4 and preterm birth associated with aberrant placentation. One possible explanation is that LTB4 elevation may potentially be a proxy for increased anti-inflammatory biomarkers, given evidence that LTB4 concentrations can remain high before and after onset of preeclampsia27. Conversely, this observation, alongside numerous null associations, may be due to underpowered sample size of this type of preterm birth in our study.Among the predictors that were linked to preterm birth associated with aberrant placentation, adaptive elastic net identified PGF and LTB4 as most predictive, both of which are protective for this type of preterm birth. PGF is a growth factor that promotes angiogenesis in part by stimulating cellular migration and recruitmentcases = 37, ncontrols = 37) observed higher concentrations of AA in plasma collected at delivery from women who delivered preterm compared to AA in plasma collected at 34 weeks gestation from women that delivered term, which is consistent with the increased risk we observed between AA and spontaneous preterm birth28. Another study evaluated differences in PGF2α and PGE2 by preterm status  and found lower levels of both in placenta and amnion samples from cases collected at delivery, but did not find significant differences in maternal plasma29. A separate case-control study  found lower levels of PGF2α measured in serum collected at the onset of active labor in preterm birth cases compared to controls30. However, another case-control study of preterm birth  did not observe notable differences in urinary concentrations of PGF2α31. Although we did not measure PGF2α, we did find higher concentrations of its metabolite 13,14DHK-PFG2α among spontaneous preterm birth cases. Ultimately there are few existing studies for comparisons, however our findings are largely consistent with current frameworks for biological mechanisms of eicosanoids.Very few studies have investigated circulating maternal eicosanoids and lipid biomarkers in association with preterm birth. One case control study of preterm birth  observed high intra-individual variability in two repeated measurements of blood eicosanoids38. In the LIFECODES cohort, we have also leveraged repeated measurements of the inflammatory biomarkers (CRP and cytokines), and observed moderate intra-individual variability9. These results provide preliminary evidence that although single time point measurements may have good utility in some study samples, repeated measurements would be advantageous when intra-individual variability is moderate or high. Future studies should consider measuring these biomarkers at multiple study visits that range across each trimester of pregnancy and conduct longitudinal analyses to determine if specific periods of gestation have greater predictive capacity.This study contains several limitations that should be discussed to improve future studies. First, our study had low numbers of cases, especially as we disaggregated to preterm birth subtypes. We may have been underpowered to observe smaller effect estimates with some of the biomarkers that we measured. The greater count of spontaneous preterm birth cases compared to preterm birth associated with aberrant placentation may have biased the results of overall preterm birth towards the biological mechanisms underlying spontaneous preterm birth. Additionally, samples were collected between 2006 and 2008, therefore biomarkers in our samples may be susceptible to degradation over long periods of storage. However, rigorous storage protocols for freezing and preserving samples partially ameliorate this problem. Furthermore, the measurement error bias associated with long storage periods would likely lead to attenuation of research findings towards null, rather than create false positives. Another limitation in our study was the cross-sectional study design and we were limited to a single time point measurement of biomarkers, therefore we were unable to evaluate time-course concentrations of individual biomarkers during pregnancy. One previous repeated measures study of select eicosanoids in adult women (n = 9) observed low intra-individual variability across eight repeated blood measurementsSince we conducted multiple comparisons, some of the single biomarker models may be at risk for false discoveries. However, the biomarkers that we discussed in depth that were selected by adaptive elastic-net circumvent the drawbacks of multiple comparisons since it evaluates all biomarkers in a single predictor matrix simultaneously rather than conducting multiple tests. We are also limited by the fact that we measured for circulating plasma or urinary biomarkers, which provides limited insight on the physiology occurring locally at the maternal-fetal interface and other target tissues. It is critical that future studies consider measuring endogenous biomarkers in multiple biological media across various time points in pregnancy. Among the biomarkers that we measured, our study focused predominantly on biomarkers measured in plasma samples. Other less invasive media such as urine, fingernail clippings, hair samples, and oral swabs can also be explored for potential predictive biomarkers. Follow up studies need to explore these additional biological media and test for their predictive capacity in observational studies. Finally, our study measured several biomarkers from the repertoire of lipid biomarkers; however we were limited in our measurement of biomarkers from the other major groups . The lack of representation of biomarkers from these other major groups may also be contributing to some degree to our observations of higher prediction capacity among lipid biomarkers compared to immune, protein oxidation, and growth factor biomarkers.Despite these limitations, our study contains several strengths to highlight. First, the study was conducted in a well-characterized birth cohort. Further, this study used high-sensitivity instrumentation to measure an extensive panel of biomarkers to assess predictors of preterm birth. Our findings provide insight on the utility of several biomarkers and groups of biomarkers to predict preterm birth. We integrated multiple statistical methods for high-dimensional data analysis that can be applied in future studies seeking to analyze a large amount of biomarkers. Another strength is that our differentiation of preterm birth subtypes provides greater clinical and physiological insight on the etiology of preterm birth. Overall our study presents new applications of statistical frameworks within biological context for future studies that seek to utilize maternal biomarkers to predict preterm birth.In conclusion, this study demonstrated that lipid biomarkers are good predictors of overall and spontaneous preterm birth. Our findings attempt to contribute a first step at prioritization of biomarker categories and eicosanoid enzymatic pathways for future studies that seek to predict preterm birth and inform precision health studies. Among eicosanoids, LOX and CYP450 products have the greatest performance of identifying overall and spontaneous preterm birth. We recommend replication studies to evaluate the consistency of our findings, and conclude that lipid biomarkers may have good utility in clinical settings to predict preterm birth.40.The LIFECODES prospective birth cohort enrolled approximately 1,600 pregnant women between 2006 and 2008 at the Brigham and Women’s Hospital in Boston, MA. Participants were between 20 and 46 years of age, recruited at less than 15 weeks gestation at the initial study visit, and attended up to 4 study visits  at which point they provided urine and blood specimens. At the initial study visit, questionnaires were administered to collect demographic and health-related information. Among participants recruited in the LIFECODES cohort, 1,181 participants were followed to term, and delivered live, single infants. This present study was cross-sectional and focused on urine and plasma samples collected at one study visit occurring between 23.1 and 28.9 weeks gestation (median = 26 weeks). The study sample included 58 women who delivered preterm (<37 weeks gestation), and 115 randomly selected women who delivered after 37 weeks gestation. Among the cases, there were 31 spontaneous preterm births and 25 preterm births associated with aberrant placentation, and 2 cases that could not be sufficiently categorized as either. This study received institutional review board (IRB) approval from the Brigham and Women’s Hospital and all participants provided written informed consent. All of the methods within this study were performed in accordance with the relevant guidelines and regulations approved by the IRB. Additional details regarding recruitment and study design can be found elsewhere9. We also measured angiogenic factors PGF and soluble fms-like tyrosine kinase-1 [sFlt-1] in plasma using the ARCHITECT immunoassays by Abbott Laboratories .Spot urine samples were collected and stored at −80 °C. Approximately 10 mL of blood was collected using ethylenediaminetetraacetic acid plasma tubes and temporarily stored at +4 °C for less than 4 hours. Afterwards, blood was centrifuged for 20 minutes and stored at −80 °C. Urine and plasma were subsequently analyzed for endogenous biomarkers. Supplemental Fig. 41. We measured for five oxidative stress biomarkers. The biomarkers total 8-isoprostane [8-IP] and 8-hydroxydeoxyguanosine [8-OHdG] were measured in urine samples by Cayman Chemical . Urine samples were hydrolyzed for deconjugation of 8-IP and then purified through affinity column chromatography prior to measurement with enzyme immunoassay. 8-OHdG was measured with direct dilution into buffer without purification using an enzyme immunoassay.The expanded panel of lipid biomarkers included 53 eicosanoids and lipid metabolites measured using a 6490 Triple Quadrupole mass spectrometer , set to a targeted multiple reaction monitoring mode. Eicosanoids were identified based on metabolite-specific fragmentation and corresponding retention time. Further details on instrument parameters and quality control can be found elsewhereo,o’-dityrosine [DY]. To quantify these markers, plasma protein was first precipitated from plasma samples and diluted with a phosphate buffer. Plasma samples were then delipidated, spiked with isotopically labeled internal standards, and hydrolyzed for 24 hours. The processed plasma samples were then analyzed for each protein oxidation marker via liquid chromatography electrospray ionization tandem mass spectrometry42.Three protein oxidative stress markers were measured in plasma samples, including 3-nitrotyrosine [NY], 3-chlorotyrosine [CY], and 5. Clinically, we defined another subset of preterm birth cases as those associated with aberrant placentation using the following criteria: (1) preterm birth resulting from the presentation of preeclampsia (encompassing onset of hypertension and proteinuria), or (2) intrauterine growth restriction  during pregnancy5.We defined overall preterm birth as delivery prior to 37 weeks gestation. Among the preterm birth cases, we categorized a subset as spontaneous preterm births based on the following clinical criteria: (1) preterm premature rupture of the membranes (PPROM), or (2) spontaneous preterm laborncases+controls = 138 for all preterm, ncases+controls = 121 for spontaneous preterm and ncases+controls = 110 for preterm birth associated with aberrant placentation. We first investigated the effects of single endogenous biomarkers on overall, spontaneous, and preterm birth associated with aberrant placentation. Measurements were log-transformed and then standardized with mean of 0 and standard deviation of 1. The effects were estimated from the model:In this study, we evaluated the associations of overall preterm birth, in addition to spontaneous preterm birth, and preterm birth associated with aberrant placentation subtypes with 65 endogenous biomarkers of interest. The subsequent analyses were restricted to individuals who had all information on covariates and biomarkers available: Y denotes an n × 1 vector of the outcome variable in separate logistic regression models where each preterm birth outcome was individually modeled to test for associations compared to term births. M is an n × 1 vector that represents the biomarker of interest. The covariate matrix Z contained age at the initial visit, race, education level, health insurance provider, and maternal BMI at the initial study visit. Age is a continuous variable and the rest of the covariates were categorical. Z further included specific gravity if M is a urinary biomarker (8-OHdG and 8-IP). To account for multiple testing and control the FDR, we applied the Benjamini-Hochberg procedure to the set of p-values obtained from Model 1 with FDR being 0.1 for each definition of outcome43. We also performed a sensitivity analysis by examining associations in unadjusted models.where Y and Z are defined in the same way as for Model 1, while M is now an n × m matrix of the biomarkers of interest with m being the number of biomarkers considered in Model 2. We applied adaptive elastic-net45, a regularized regression approach, to each of the three outcome definitions separately using the R package gcdnet. The estimates of the p × 1 vector β = T were obtained by solving:One of our study goals was to determine which of the 65 biomarkers were contributing most to associations with preterm birth in a multiple biomarker model:46 and ridge47 type penalty to perform variable selection with a correlated set of candidate predictors. The function h is an assumed loss function, and the the adaptive weights λ2) take values on a grid from 0 to 2, in increments of 0.1. Furthermore, we forced the covariates Z to always be included in the model. For each fixed λ2, the function automatically computes the solutions for a fine grid of λ1’s, the λ1 and λ2 that yielded the smallest 10-fold cross-validated misclassification error and used their corresponding coefficient estimates as adaptive weights.which uses both lasso41, or if they are parent compounds  DNAds Table . To inveL(β) is the likelihood function for Model 2, q is the total number of groups considered (in this case q = 5), pk is the number of predictors in group k, and ∥1 and ∥2 are L1 and L2 norm, respectively. The mixing parameter α and tuning parameter λ together controls both “groupwise sparsity” and “within group sparsity” of non-null biomarkers. We set α to be 0.95, which assumes strong overall sparsity but encourages grouping48. In a secondary sparse-group lasso implementation, we split the major group of lipid damage markers into five subgroups, which included groupings for the three enzymatic pathways, lipid parent compounds, and 8-IP on its own (q = 9). Since current R implementation of sparse-group lasso does not support the specification of non-penalized coefficients, we treated the group of covariates as an additional group48. The relative importance of the associations was evaluated based on the sequence of the path in which each group exits the model as the tuning parameter λ increased (the group exiting last being the most important).where 49 to achieve balance between cases and controls, which may improve the prediction of minority class (in this case preterm birth). We used three different prediction algorithms using: (1) logistic regression, (2) adaptive elastic-net, and (3) random forest. First, we fitted a standard logistic regression model (Model 2) with the coefficients being estimated by maximum likelihood to the resampled training data for all biomarkers organized by each major group, and each eicosanoid enzymatic pathway. This method serves as a benchmark for evaluating the performance in prediction. The group of lipid damage biomarkers  was excluded for this approach because the maximum likelihood estimates obtained from logistic regression were unstable due to the high dimension of predictors. Second, we trained the model by applying adaptive elastic-net to the training set for each major group of biomarkers, the process of which was the same as what was done on the whole data set. Building on this framework, we also applied the same adaptive elastic-net method to the training set where we focused solely on the lipid biomarkers differentiating the three enzymatic pathways and the parent compounds. Finally, going beyond parametric regression methods, we trained the model using random forest for all groups considered, where the number of variables randomly sampled at each split for the random forest method was determined using 10-fold cross validation. The logistic regression prediction approach is limited and becomes unstable in the presence of multicollinearity and high-dimensionality of covariates and predictors. The adaptive elastic-net prediction method has improved performance in the presence of multi-collinearity, however it is unable to account for non-linear relationships between predictors and outcome variables. Finally, the random forest prediction method is able to account for non-linear relationships and interactions among predictors.We evaluated the performance of all and grouped endogenous biomarkers in predicting preterm birth. Subjects were randomly divided into training set and validation set with a ratio of 70:30 for the respective comparison of overall, spontaneous, and preterm birth associated with aberrant placentation vs. term deliveries. The training set was resampled using the Synthetic Minority Over-sampling TechniqueFor all three prediction approaches, we obtained the predicted probability of the outcome of interest for each subject in the validation set using the optimal parameters determined by the training set. Subjects were classified as cases if their predicted probabilities were greater than 0.5 and controls otherwise. We calculated the misclassification error, sensitivity and specificity, the latter two of which are defined as the probability of correctly identifying the participants with and without preterm delivery, respectively. Over fitting can be a possible concern in the logistic regression prediction method; however over fitting is not a major concern in both adaptive elastic-net and random forest prediction methods. We also examined the ROC curve and computed the AUC to assess the performance of the final model.This study received institutional review board approval from the Brigham and Women’s Hospital and all participants provided written informed consent.Supplementary material


Mycobacterium tuberculosis one of the most successful pathogens to survive for long durations within human cells. In order to survive against host defenses, M. tuberculosis modulates host cell signaling. It employs many proteins to achieve this and the Mce proteins are emerging as one group that play a role in host cell signaling in addition to their primary role as lipid/sterol transporters. Mce proteins belong to the conserved Mce/MlaD superfamily ubiquitous in diderm bacteria and chloroplasts. In mycobacteria, mce operons, encode for six different Mce proteins that assemble with inner membrane permeases into complexes that span across the mycobacterial cell wall. Their involvement in signaling modulation is varied and they have been shown to bind ERK1/2 to alter host cytokine expression; eEF1A1 to promote host cell proliferation and integrins for host cell adherence and entry. Recently, structures of prokaryotic Mce/MlaD proteins have been determined, giving an insight into the conserved domain. In this mini-review, we discuss current evidence for the role of mycobacterial Mce proteins in host cell signaling and structural characteristics of the protein-protein interactions coordinated by the human proteins to modulate the host signaling.Tuberculosis continues to be the main cause for mortality by an infectious agent, making  Mycobacterium tuberculosis, has sophisticated mechanisms to promote its own survival within humans. The tussle of bacterial survival vs. disease progression is governed by dynamic interactions between M. tuberculosis and host cells. Inhaled aerosolized bacteria primarily encounter alveolar macrophages and their ability to persist in an antimicrobial environment is critical for establishing an infection A-F] and two permease proteins which assemble with an ATPase, termed MceG, to form assemblies resembling ABC-transporters that reside in the extracellular membrane  protein assemblies from membrane . These amembrane . The MceEscherichia coli , a gram-negative outer membrane Mce protein with seven tandem Mce domains from Vibrio parahaemolyticus has been shown to bind both host lipids and proteins to enable host cell adhesion alluding to the role of Mce proteins in manipulating host cell signaling  signaling pathway , reveals a binding interaction that might be employed by Mce2E and Mce3E. PEA-15 binds to ERK1/2 preventing its nuclear translocation similar to the mechanism exhibited by Mce2E and Mce3E. ERK2 consists of two domains: a smaller N-terminal domain that consists of mainly -strands and sits on the C-terminal domain that is comprised of mostly alpha helices. The C-terminal domain contains the activation loop which is dually phosphorylated for activation . PEA-15 M. tuberculosis disrupting host cell degradation pathways has been documented previously with the PtpA protein (Wang et al., In addition to its suppression of macrophage immune responses, Mce2E has been shown to promote epithelial cell proliferation (Qiang et al., eEF1A1 is expressed in the majority of cell types and in its primary function when GTP-bound, it binds aminoacylated-tRNA delivering it to the A site of ribosomes during polypeptide chain elongation in translation (Mateyak and Kinzy, Beyond the canonical role of eEF1A1 in protein translation, it has several non-canonical roles including proteolysis, protein nuclear export, cytoskeleton organization, and apoptosis (Mateyak and Kinzy, M. tuberculosis appears to employ receptor binding as another method to utilize host signaling for its advantage. Zhang et al. demonstrated that Mce3C, present on the surface of mycobacteria, binds to ß2 integrin promoting adherence and entry into macrophages. The binding is RGD motif dependent and leads to integrin dependent SFKs-Syk-Vav-Rho-ROCK signal transduction inducing local actin rearrangements thus promoting mycobacterial entry. The RGD motif of Mce3C has a high affinity for the leucocyte-restricted ß2 integrin binding to its I-like domain (Pathogens are known to exploit receptor-mediated signaling to avoid antimicrobial strategies of host cells including to prevent or abolish phagocytosis. e domain . Mutatioe domain .Leptospira interrogans strain Lai. It binds to α5ß1, αvß3, and ß2 integrins to promote pathogen adhesion and invasiveness (Zhang et al., The RGD motif is a well-documented ligand binding motif of integrins  and one-M. tuberculosis, eight contain RGD motifs with a total of 10 (Mce1E and Mce2E contain two each; Of the 24 Mce proteins in M. tuberculosis to different host signaling proteins highlights their role beyond lipid transport. Thus far, they have been implicated in altering cytokine expression through interaction with ERK1/2; promoting cell proliferation through preventing proteasomal degradation of eEF1A1 and enabling cell adherence and entry through interactions with integrins. The current research is limited to a few studies encompassing three out of the 24 Mce proteins in M. tuberculosis. When all 24 are considered in the context of their membrane complexes their effect on host cell signaling could be vast and varied. There is some evidence to suggest that the four mce operons are expressed at different times during the infection life-cycle (Joshi et al., M. tuberculosis proteins such as Mce, ZmpI, and PtpA interacting with many signaling pathways is key to understanding the sophistication the pathogen employs to survive.The binding of Mce proteins from KF and VD conceptualized the manuscript. KF, CW, and VD wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this meta-analysis was to compare clavicle hook plates versus distal clavicle locking plates for the treatment of Neer type II distal clavicle fractures.PubMed (1996 to January 2019), Embase (1980 to January 2019), Web of Science (1990 to January 2019), the Cochrane Library (January 2019), and the China National Knowledge Infrastructure (January 2019) were systematically searched without language restrictions for literature retrieval. The Constant-Murley shoulder joint function score at 3 and 6 months after the operation and the postoperative complications after the operation  were the outcomes. Stata 12.0 was used for the meta-analysis.P < 0.05). There were fewer cases of shoulder joint pain and restricted shoulder abduction range of motion in the distal locking plate group, and the difference was statistically significant (P < 0.05). There were no statistically significant differences in fracture delay healing and subacromial impingement between the two groups (P > 0.05).A total of 9 clinical trials involving 446 patients were finally included in this meta-analysis. The results showed that the improvement in the Constant-Murley shoulder joint function score in the distal locking plate group was better than that in the clavicle hook plate group at 3 and 6 months after the operation (Compared with the clavicular hook plate, the distal clavicle locking plate for the treatment of Neer type II distal clavicle fractures is associated with better shoulder function recovery and fewer complications related to pain and abduction restriction. Clavicular fractures are common and typically occur in young patients, leading to a burden for this active population , 2. ClavStrategies for the treatment of clavicular fractures include coracoclavicular fixation (sutures such as a tight rope or endobutton and screw) and fracture fixation devices  , 9. ClavHowever, there have been no systematic, quantitative evaluations comparing the two methods. In this systematic review and meta-analysis, we included relevant studies to compare the clinical outcomes of clavicular hook plates and clavicular locking plates in individuals with clavicular fractures to provide some evidence for clinical decision making.We performed a meta-analysis that compared the clinical efficacy and safety of clavicular hook plates and clavicular locking plates for the treatment of Neer type II distal clavicle fractures.This meta-analysis was conducted based on the recommendations in the Cochrane Handbook for Systematic Reviews of Interventions and was written in accordance with the PRISMA  checklist . The metPubMed (1996 to January 2019), Embase (1980 to January 2019), Web of Science (1990 to January 2019), the Cochrane Library (January 2019), and the China National Knowledge Infrastructure (January 2019) were systematically searched. The references of all randomized controlled trials (RCTs) were searched for additional studies related to Neer type II distal clavicle fractures. The keywords used were “distal clavicle fractures,” “clavicle fractures,” “clavicle hook plate,” and “distal clavicle locking plate” in conjunction with the Boolean operators “AND” or “OR”. The two authors independently reviewed the titles and abstracts of the articles to exclude significantly unrelated studies. The search procedure is presented in Fig. Studies were eligible for the meta-analysis if they met the following criteria: (1) participants: patients with distal clavicle fractures; (2) intervention: patients receiving a clavicle hook plate in the study group and distal clavicle locking plate in the study group; (3) outcomes: Constant-Murley scores at 3 months and 6 months, the occurrence of shoulder pain, restricted shoulder abduction range of motion cases, delayed union and acromion impingement syndrome; and (4) study design: RCT. Studies that did not include the outcome measures of interest were excluded. Letters, abstracts, comments, editorials, and studies without the full text available were also excluded. Disputes were resolved through discussion or the third reviewer was consulted.Two authors independently extracted the available data from the included studies. The data included the first author, publication date, number of patients, location of the fracture, incision type, fracture type, number of female patients, patient’s age, follow-up duration, outcomes, and study type. The primary outcome included the Constant-Murley scores at 3 months and 6 months. The secondary outcomes included the occurrence of shoulder pain, restricted shoulder abduction range of motion cases, delayed union, and acromion impingement syndrome. All extracted data were summarized in a pre-designed Microsoft Excel worksheet. Disagreements were resolved by discussion to reach a consensus.Two authors (Liang Li and Tian-yan Li) independently evaluated the risk of bias of the included studies according to the Cochrane Handbook, which considered the following items: “random sequence generation,” “allocation concealment,” “blinding of participants and personnel,” “blinding of outcomes assessors,” “incomplete outcome data,” “selective outcome reporting,” and “other bias” . The metP value and I2 statistic using the chi-squared test. If I2 > 50% or P < 0.05, the studies were considered to demonstrate significant heterogeneity, and a random-effects model was used; otherwise, a fixed-effects model was used. This meta-analysis also used a funnel plot of the urinary tract infection data to independently assess publication bias.The current meta-analysis was conducted using Stata 12.0 . For the continuous outcomes, the mean difference (MD) with a 95% confidence interval (CI) was used. For the discontinuous outcomes, relative risk (RR) with a 95% CI was used. The statistical heterogeneity was assessed by the A total of 454 studies were identified through the initial search, and 226 papers were excluded because they were duplicates. In the next stage, 216 papers were excluded after the titles and abstracts were read. Therefore, 9 RCTs , 13–19 wThe baseline characteristics of the included studies are summarized in Table Figures P = 0.000; Fig. I2 = 36.0%).Seven trials involving 306 participants reported Constant-Murley scores at 3 months. Compared with the clavicle hook plate, the distal clavicle locking plate was associated with higher Constant-Murley scores at 3 months .Seven trials involving 331 participants reported Constant-Murley scores at 6 months. Compared with the clavicle hook plate, the distal clavicle locking plate was associated with higher Constant-Murley scores at 6 months .Six trials involving 289 participants reported the occurrence of shoulder pain. Compared with the clavicle hook plate, the distal clavicle locking plate was associated with a decrease in the occurrence of shoulder pain .Four trials involving 166 participants reported a restricted shoulder abduction range of motion cases. Compared with the clavicle hook plate, the distal clavicle locking plate was associated with a decrease in the number of restricted shoulder abduction range of motion cases .Six trials involving 292 participants reported the occurrence of delayed union. There was no statistically significant difference between the clavicle hook plate and distal clavicle locking plate groups in terms of the occurrence of delayed union .Six trials involving 168 participants reported the occurrence of acromion impingement syndrome. There was no statistically significant difference between the clavicle hook plate and distal clavicle locking plate groups in terms of the occurrence of acromion impingement syndrome (RR, 0.95; 95% CI, 0.38 to 2.33; A sensitivity analysis was performed by omitting included studies sequentially, and the results suggested that after removing each study sequentially, the overall effect size was not changed Fig. .Fig. 10P = 0.69, Fig. P = 0.73, Fig. For the meta-analysis of the Constant-Murley scores at 3 months, there was no evidence of publication bias according to an inspection of the funnel plot Fig.  and formCompared with the clavicle hook plate, the distal clavicle locking plate was associated with increased Constant-Murley scores at 3 months and 6 months. In addition, the distal clavicle locking plate was associated with a decrease in the occurrence of shoulder pain and the number of restricted shoulder abduction range of motion cases. There was no significant difference between the clavicle hook plate and distal clavicle locking plate groups in terms of the occurrence of delayed union and the occurrence of acromion impingement syndrome. We performed a sensitivity analysis and found that after removing each study sequentially, the overall effect size was not changed.Only one previous systematic review and network meta-analysis comparing clavicle hook plates and distal clavicle locking plates have been published . The majIn this meta-analysis, we used the Constant-Murley scores at 3 months as the primary outcome. The results showed that compared with the clavicle hook plate, the distal clavicle locking plate was associated with increased Constant-Murley scores at 3 months and 6 months. Our results were compatible with the results of previous studies , 21. ZhaThe treatment of Neer type II distal clavicle fractures is a controversial topic. Several studies have recommended open reduction and internal fixation for Neer type II distal clavicle fractures because these fractures tend to displace and are associated with a higher risk of nonunion compared with other clavicle fracture types. We compared the occurrence of delayed union between the clavicle hook plate and distal clavicle locking plate groups. We found that there was no significant difference between the occurrence of delayed union between the clavicle hook plate and distal clavicle locking plate groups.We then compared the occurrence of shoulder pain and the restricted shoulder abduction range of motion cases between the clavicle hook plate and distal clavicle locking plate groups. Compared with the clavicle hook plate, the distal clavicle locking plate was associated with a decrease in the occurrence of shoulder pain and a restricted shoulder abduction range of motion cases.Some limitations of this study should be noted. First, the small sample size might have affected the significant difference between the two surgical procedures. Second, most of the included studies originated from China, and more RCTs published in other countries should be conducted to confirm this conclusion. Third, our study ignored the diversity of diagnostic criteria and etiology of the disease, and further research is needed to determine whether these conclusions apply to patients with the various other types of distal clavicle fractures.Compared with the clavicular hook plate, the distal clavicle locking plate for the treatment of Neer type II distal clavicle fractures is associated with better shoulder function recovery and fewer complications related to pain and abduction restriction. In view of the heterogeneity and different follow-up times, whether these conclusions are applicable should be further assessed in future studies.


Genome-wide association studies (GWAS) and fine mapping studies in autoimmune diseases have identified thousands of genetic variants, the majority of which are located in non-protein-coding enhancer regions. Enhancers function within the context of the three-dimensional (3D) genome to form long-range DNA looping events with target gene promoters that spatially and temporally regulate gene expression. Investigating the functional significance of GWAS variants in the context of the 3D genome is essential for mechanistic understanding of these variants and how they influence disease pathology by altering DNA looping between enhancers and the target gene promoters they regulate. In this review, we discuss the functional complexity of the 3D genome and the technological approaches used to characterize DNA looping events. We then highlight examples from the literature that illustrate how functional mapping of the 3D genome can assist in defining mechanisms that influence pathogenic gene expression. We conclude by highlighting future advances necessary to fully integrate 3D genome analyses into the functional workup of GWAS variants in the continuing effort to improve the health of patients with autoimmune diseases. Genome-wide association studies (GWAS) have significantly advanced the identification of variants associated with complex genetic diseases, including autoimmune diseases , 2. GWASTo demystify the function of non-coding DNA in chromatin regulation and gene expression, several large-scale collaborative efforts (Encyclopedia of DNA Elements (ENCODE) Project, National Institutes of Health (NIH) Roadmap Epigenomics Mapping Consortium, and International Human Epigenome Consortium (IHEC)) have successfully mapped the locations of regulatory sequences that bind over 400 transcription factors and histone post-translational modifications that mark enhancers, promoters, repressors, and insulator regions in a large variety of cell lines and primary cells –8. ColleEnhancer elements are short DNA sequences ~ 50–1500 bp) that bind transcription factors leading to the expression of a gene . It is e0–1500 bpIn this review, we discuss the functional complexity of the 3D genome, contrast the various aspects of how the 3D genome is measured, and provide specific examples of how knowledge of the 3D genome has helped decipher GWAS results. We conclude by highlighting future advancements needed to generalize 3D genome data into the routine analysis of GWAS-associated risk variants.The human genome is organized into complex layers of intricate folds and loops that allow for proper gene expression regulation while fitting roughly three meters of histone-wrapped DNA into an interphase nucleus averaging 6 μm in diameter , 14, 15.Characterizing the complex layers of organization that occur within the 3D genome and the regulatory mechanisms that dictate them have provided a framework from which current explorations of gene expression regulation are often based. Approximately 90% of identified enhancer–promoter loops occur within the CTCF-CTCF–cohesin boundaries of TADs and insulated neighborhoods . As repoThe majority of techniques used to investigate the 3D genome are derivatives of the original chromatin conformation capture (3C) method, which uses a process called proximity ligation to capture interactions between two sequences of DNA that are in 3D proximity but are separated by linear distance  and H19 gene loci alters expression of the non-coding RNA, H19 . Fo. Fo21]. Single cell Hi-C was first reported in 2013 to explore the cell-to-cell variability of chromatin structures using a single copy X-chromosome model in isolated mouse nuclei . More reProtein-mediated genome-wide methodologies Table  include 3D genome-wide exploration generates tremendous amounts of sequencing data that require advanced algorithms and pipelines for processing, visualizing, and interpreting the functional significance of these 3D features. Fortunately, several robust software packages are publicly available and more are in development. Each algorithm uses different alignment strategies and filtering criteria to generate heatmaps based on interaction frequencies , 53 or lTNFAIP3) gene promoter [TNFAIP3 is a critical negative regulator of pro-inflammatory nuclear factor kappa B (NF-κB) signaling implicated in many autoimmune diseases, and therefore a suspected target gene of the identified enhancer [TNFAIP3 gene expression by bringing transcription factors, including NF-κB, to the TNFAIP3 promoter region via long-range enhancer–promoter interactions [TNFAIP3 promoter, effectively suppressing TNFAIP3 expression [Autoimmune diseases, like most complex genetic diseases, result from the collective influence of multiple genetic variants on gene expression and responses to potentially damaging environmental conditions . Investipromoter , 56. TNFenhancer –57. Funcractions . ImportaOLIG3) and TNFAIP3. In contrast to the well characterized role of TNFAIP3 in regulating inflammatory signaling pathways, OLIG3 is an important regulator of neuronal development and has no established role in the immune system [TNFAIP3 was the likely target of this enhancer. To test this hypothesis, Capture Hi-C studies in both B and T cells from patients with rheumatoid arthritis were performed. Interestingly, these studies revealed that chromatin loops formed not only between the enhancer and the downstream promoter of TNFAIP3, but also with the promoters of interleukin 20 receptor subunit alpha (IL20RA) and interferon gamma receptor 1 (IFNGR1) located 180 kb upstream [IL20RA gene expression [OLIG3 promoter, effectively eliminating this gene from further consideration. Together, these studies demonstrate the utility of targeted 3D genome applications to test and refine hypotheses regarding loop formation with enhancers harboring GWAS risk variants and their impact on gene expression.3D genome analysis is also a powerful method for identifying unsuspected candidate genes whose expression could be altered by risk variants in enhancers. For example, an enhancer harboring GWAS risk variants for rheumatoid arthritis was identified between oligodendrocyte transcription factor 3 , resides [TAL1 expression. Disrupting this CTCF boundary allows the promoter of a nearby gene, STIL centriolar assembly protein (STIL), to reposition near TAL1 and activate expression [Variants that disrupt anchor protein motifs, such as CTCF motifs that define TAD and insulated neighborhood boundaries, also have the potential to disrupt gene expression regulation by permitting aberrant boundary formation and allowing unrestricted enhancer activation of genes normally excluded from the neighborhood Fig. . An exam resides . Importapression . This ispression , 61, 62.ORMDL3), a gene that facilitates cytokine production in the lung [3D genome technologies that typically require tens of millions of input cells have largely limited investigations into the potential implications of disrupting CTCF boundaries in primary immune cells involved in autoimmune disease pathogenesis. However, a recent report using 4C with NGS demonstrated that two asthma risk variants at the chromosome 17q21 locus, rs4065275 and rs12936231, individually altered CTCF binding motifs in CD4+ and CD8+ T cells . In boththe lung . This stAnalysis of 3D chromatin topology is an essential component for a complete mechanistic understanding of how genetic variants associated with complex human disease drive disease pathogenesis. As large-scale 3D chromatin technologies improve, we anticipate many of their current shortcomings will dissipate. In particular, we look forward to improvements in analytical methods that would facilitate truly quantitative comparisons of relative loop frequencies and enhancer–promoter interactions between different cell types and conditions. This, combined with reductions in chromatin input requirements such that small numbers of primary cells could be analyzed, would scale the potential of 3D chromatin studies in a manner that is now common for transcriptome studies.The recently established 4D Nucleome Project supported by the NIH Common Fund  is charg


KCNJ13 are associated with two retinal disorders; Leber congenital amaurosis (LCA) and snowflake vitreoretinal degeneration (SVD). We describe a novel fibrovascular proliferation in the retina of two affected members of a KCNJ13-related LCA family with a homozygous c.458C > T, p.(Thr153Ile) missense mutation. Optical coherence tomography retinal imaging of the kcnj13 mutant zebrafish  revealed a late onset retinal degeneration at 12 months, with retinal thinning and associated retinovascular changes, including increased vessel calibre and vitreous deposits. Both human and zebrafish variants are missense and located within the conserved transmembrane M2 protein domain, suggesting that disruption of this region may contribute to retinovascular changes as an additional feature to the previously described LCA phenotype. Close monitoring of other patients with similar mutations may be required to minimise the ensuing retinal damage.Mutations in  •KCNJ13 c.458C>T, p.(Thr153Ile) mutations were examined for retinovascular abnormalities.Patients with •KCNJ13-Leber congenital amaurosis.A novel fibrovascular proliferation was observed in the retina of two siblings with •kcnj13 mutant zebrafish (obelixtd15) showed a retinal degeneration with associated retinovascular changes.•Both human and zebrafish variants are missense and located within the transmembrane M2 protein domain.•KCNJ13-related retinal disease.This retinovascular complication should be considered within the clinical spectrum of  LCA is #614186) . It is cutations . SVD is utations . The visobelix (obetd15) zebrafish mutant, generated through ENU mutagenesis, harbors a missense mutation  in kcnj13, which affects the transmembrane region abolishing K+ conductance by disrupting K+ permeation through the channel  with Optos imaging, as part of routine clinical care.The study was approved by the local research ethics committee, and all investigations were conducted in accordance with the principles of the Declaration of Helsinki; informed consent was obtained from all participating individuals. Molecular genetic testing on genomic DNA extracted from blood using an LCA next-generation sequencing (NGS) panel including 2.2obetd15 zebrafish were generated by natural pair-wise matings of genotyped homozygous or heterozygous fish and raised at 28.5 °C on a 14 h light/10 h dark cycle in the UCL zebrafish facility. Zebrafish were maintained according to local UCL and UK Home Office regulations for the care and use of laboratory animals under the Animals Scientific Procedures Act at the UCL Institute of Ophthalmology animal facility. UCL Animal Welfare and Ethical Review Body approved all procedures for experimental protocols, in addition to the UK Home Office (License no. PPL PC916FDE7). All zebrafish experimentation followed the ARRIVE guidelines for animal research.Wild-type AB and 2.3obetd15 retinas were scanned using the Bioptigen Envisu R2200 Spectral Domain Ophthalmic Imaging System  as described  in KCNJ13 , Fig. 1.KCNJ13-LCA . The initial appearances of the retinas in the two brothers looked fairly typical of LCA with extensive RPE changes in both the macula and retinal peripheries with progressive pigment migration and vessel attenuation  and two intravitreal injections of avastin. Following a left vitreous haemorrhage, the patient had a pars plana vitrectomy with secondary complications of cataracts requiring phacoemulsification followed by uveitic glaucoma resulting in posterior capsule opacification and membranectomy. Patient A-2 also developed retinal neovascularization and right vitreous haemorrhage requiring extensive PRP. FFA was performed to investigate for ongoing retinal neovascularization and early and late leakage is seen in both patient retinas d–f. FFA KCNJ13-LCA, where affected patients are between 10 and 34 years, no previous reports of retinovascular changes have been described , suggesting that the mutant vessels were wider for a given distance from the optic nerve compared to wild-type controls. OCT imaging of heterozygous obetd15 zebrafish showed retinal vasculature comparable to that of wild-type fish  and perhaps dependent on the specific nature and position of the mutation and its impact on protein function.We have described here retinovascular abnormalities in two siblings (A-1 and A-2) with homozygous c.458C > T, p.(Thr153Ile) eviously . These aKCNJ13 model zebrafish, obetd15, has been previously described  mutation did not exhibit any clinically significant retinal vascular abnormalities. This could mean that the retinovascular abnormalities might manifest itself from teenage years onwards or there could be other genes that play a role in the expression of this phenotype. For affected members of the family who have the same mutation but no vascular changes, regular monitoring would be recommended as they are not yet of the same age and the onset may be forthcoming.The two other younger siblings from a different family (B-1 and B-2) with the same KCNJ13 missense mutations. Further experimentation will be necessary to determine any involvement of Kir7.1 in vascular growth and function.In summary, we have reported novel retinovascular findings in both human LCA patients and a corresponding zebrafish mutant, associated with None.


Gillespie syndrome is a rare, congenital, neurological disorder characterized by the association of partial bilateral aniridia, non-progressive cerebellar ataxia and intellectual disability. Homozygous and heterozygous pathogenic variants of the ITPR1 gene encoding an inositol 1, 4, 5- triphosphate- responsive calcium channel have been identified in 13 patients recently. There have been 22 cases reported in the literature by 2016, mostly from the western hemisphere with none reported from Sri Lanka.A 10-year-old girl born to healthy non-consanguineous parents with delayed development is described. She started walking unaided by 9 years with a significantly unsteady gait and her speech was similarly delayed. Physical examination revealed multiple cerebellar signs. Slit lamp examination of eyes revealed bilateral partial aniridia. Magnetic resonance imaging of brain at the age of 10 years revealed cerebellar (mainly vermian) hypoplasia. Genetic testing confirmed the clinical suspicion and demonstrated a heterozygous pathogenic variant c.7786_7788delAAG p.(Lys2596del) in the ITPR1 gene.The report of this child with molecular confirmation of Gillespie syndrome highlights the need for careful evaluation of ophthalmological and neurological features in patients that enables correct clinical diagnosis. The availability of genetic testing enables more accurate counseling of the parents and patients regarding recurrence risks to other family members. Gillespie syndrome, also known as, aniridia-cerebellar ataxia-intellectual disability syndrome, is a rare form of congenital dysautonomia characterized by non-progressive cerebellar ataxia, partial aniridia, and intellectual impairment . Since iThe proband is a 10-year-old Sri Lankan girl of Sinhala ethnicity born to healthy non-consanguineous parents with two other older, healthy sons. She had an uneventful pregnancy and birth history and there was no family history of ophthalmological or neurological diseases. Although her parents noted delayed development, they had not sought medical attention for this. She started walking unaided at around 9 years and continues to have a significantly unsteady gait. Her hearing was not affected but speech was delayed (first word at 3 years and currently speaking 3–4-word complex sentences). Her parents reported photophobia since infancy.On examination aged 10 years, her height, weight and occipito-frontal circumference were 132 cm (10th–25th centile), 26 kg (25th–50th centile) and 51 cm (25th–50th centile) respectively. She had no dysmorphic features. A pigmented macule with a serpiginous border (22 cm × 10 cm), which may represent a blaschcoid pattern suggestive of a cutaneous mosaicism, was noted on her right thigh  of the brain revealed cerebellar hypoplasia especially affecting the vermis and white matter changes Fig. . CervicaGillespie syndrome was first described in two adult siblings who had the triad of partial aniridia, cerebellar ataxia and oligophrenia . The synThis diagnosis needs to be considered in infants with aniridia associated with hypotonia. Bilateral partial aniridia is consistently seen in Gillespie syndrome. It has a characteristic scalloped pupillary border, iris strands with regularly spaced attachments to the anterior lens with some reports of persistence of the pupillary membrane. Complete aniridia, especially when associated with foveal hypoplasia, cataracts, corneal opacification with only rare extraocular involvement is usually inherited as an autosomal dominant trait; mutations of the Paired box protein 6 (PAX6) gene are commonly implicated . ContiguThe second major diagnostic criterion for Gillespie syndrome is the involvement of the brain and in particular, the cerebellum. In early life, the majority of cases appear to have hypotonia with marked delay in gross and fine motor milestones being commonly reported in later infancy and childhood . CerebelFacial dysmorphism , pectoraITPR1 gene pathogenic variants are the underlying genetic defects for autosomal recessive and dominant Gillespie syndrome respectively. The ITPR1 gene encodes one of the three subtypes of the inositol triphosphate (IP3)-receptor family that form Ca2+ release channels especially in the endoplasmic reticulum. It is also expressed in the nervous system especially the Purkinje cells of the cerebellum [The genetic basis of this syndrome was defined with identification of pathogenic variants in the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) gene on chromosome 3p26 [rebellum . The recClinical diagnosis of Gillespie syndrome can be made following identification of its two cardinal features of cerebellar ataxia and partial aniridia. All cases of aniridia require careful clinical evaluation for additional ocular and extra-ocular features. Once suspected, targeted genetic testing will confirm the diagnosis and enable counseling regarding prognosis and risk of the disease in other family members. As the parents were not planning more family, the confirmation of a de novo pathogenic variant or inheritance from an unaffected parent was not pursued. This may be required if her clinically unaffected brothers wish to ascertain their status once they are older. Although this child’s testing was performed free of charge, the costs of genetic testing remain a barrier for accurate genetic diagnosis in patients from developing countries.