Sexually dimorphic effects of pexidartinib on nerve injury-induced neuropathic pain in mice.

It is well-established that spinal microglia and peripheral macrophages play critical roles in the etiology of neuropathic pain; however, growing evidence suggests sex differences in pain hypersensitivity owing to microglia and macrophages. Therefore, it is crucial to understand sex- and androgen-dependent characteristics of pain-related myeloid cells in mice with nerve injury-induced neuropathic pain. To deplete microglia and macrophages, pexidartinib (PLX3397), an inhibitor of the colony-stimulating factor 1 receptor, was orally administered, and mice were subjected to partial sciatic nerve ligation (PSL). Following PSL induction, healthy male and female mice and male gonadectomized (GDX) mice exhibited similar levels of spinal microglial activation, peripheral macrophage accumulation, and mechanical allodynia. Treatment with PLX3397 significantly suppressed mechanical allodynia in normal males; this was not observed in female and GDX male mice. Sex- and androgen-dependent differences in the PLX3397-mediated preventive effects were observed on spinal microglia and dorsal root ganglia (DRG) macrophages, as well as in expression patterns of pain-related inflammatory mediators in these cells. Conversely, no sex- or androgen-dependent differences were detected in sciatic nerve macrophages, and inhibition of peripheral CC-chemokine receptor 5 prevented neuropathic pain in both sexes. Collectively, these findings demonstrate the presence of considerable sex- and androgen-dependent differences in the etiology of neuropathic pain in spinal microglia and DRG macrophages but not in sciatic nerve macrophages. Given that the mechanisms of neuropathic pain may differ among experimental models and clinical conditions, accumulating several lines of evidence is crucial to comprehensively clarifying the sex-dependent regulatory mechanisms of pain.

Early-onset herpes simplex encephalitis type 1 triggered by COVID-19 disease: A case report.

Coronavirus disease 2019 (COVID-19) causes a systemic inflammatory response and a temporary immunosuppression of hosts. Several reports have showed that reactivation of herpes simplex virus type 1 (HSV-1) is strongly associated with COVID-19. We present a case of a 66-year-old female, who developed HSV-1 encephalitis, showing impaired consciousness and typical MRI findings such as hyperintense lesions in the temporal lobe, insular cortices, bilateral medial frontal lobe on diffusion-weighted imaging, 7 days after the onset of COVID-19 symptoms. The number of cases of encephalitis in patients with COVID-19 is increasing. However, there has been limited reports of HSV-1 encephalitis following COVID-19, especially for cases with an interval of 7 days or less from the onset of COVID-19 symptoms to the onset of HSV-1 encephalitis. Our case highlights the importance of considering HSV-1 encephalitis in the differential when managing a patient with COVID-19-associated neurologic complications, even if it is in the early stages of COVID-19.

Elucidation of HHEX in pancreatic endoderm differentiation using a human iPSC differentiation model.

For pluripotent stem cell (PSC)-based regenerative therapy against diabetes, the differentiation efficiency to pancreatic lineage cells needs to be improved based on the mechanistic understanding of pancreatic differentiation. Here, we aimed to elucidate the molecular mechanisms underlying pancreatic endoderm differentiation by searching for factors that regulate a crucial pancreatic endoderm marker gene, NKX6.1. Unbiasedly screening an siRNA knockdown library, we identified a candidate transcription factor, HHEX. HHEX knockdown suppressed the expression of another pancreatic endoderm marker gene, PTF1A, as well as NKX6.1, independently of PDX1, a known regulator of NKX6.1 expression. In contrast, the overexpression of HHEX upregulated the expressions of NKX6.1 and PTF1A. RNA-seq analysis showed decreased expressions of several genes related to pancreatic development, such as NKX6.1, PTF1A, ONECUT1 and ONECUT3, in HHEX knockdown pancreatic endoderm. These results suggest that HHEX plays a key role in pancreatic endoderm differentiation.

Racial difference in the association between non-alcoholic fatty liver disease and incident type 2 diabetes: findings from the CARDIA study.

AIMS/HYPOTHESIS: Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are prevalent diseases of metabolic origin. We examined the association between NAFLD and the development of type 2 diabetes among non-Asian adults, and whether the association differs by race. METHODS: We analysed data from the Coronary Artery Risk Development in Young Adults (CARDIA) study, a population-based prospective cohort study. Participants underwent non-contrast abdominal computed tomography (CT) at baseline (2010-2011) and assessment of glucose measures at the follow-up exam (2015-2016). NAFLD was defined as liver attenuation ≤51 Hounsfield units on CT images after exclusion for other liver fat causes. Race was self-reported. We used targeted maximum likelihood estimation (TMLE) with machine-learning algorithms to estimate difference in type 2 diabetes risk between the NAFLD and non-NAFLD groups. RESULTS: Of the 1995 participants without type 2 diabetes at baseline (mean age±SD, 50.0±3.6 years; 59% women; 55.0% White and 45.0% Black), 21.7% of White and 16.8% of Black participants had NAFLD at baseline, and 3.7% of White and 8.0% of Black participants developed type 2 diabetes at follow up. After multivariable adjustment, risk difference for type 2 diabetes associated with NAFLD vs no NAFLD was 4.1% (95% CI 0.3%, 7.9%) among White participants and -1.9% (95% CI -5.7%, 2.0%) in Black participants. CONCLUSIONS/INTERPRETATION: NAFLD was associated with a higher risk of type 2 diabetes among White participants but not among Black participants. This finding suggests that the effect of liver fat on impaired glucose metabolism may be smaller in Black than in White individuals.

Targeted DNA Methylation in Mouse Early Embryos.

In germ cell lines, including early preimplantation embryos, centromeres and pericentromeres are known to show a marked hypomethylation pattern compared to somatic cells. Elucidation of the biological function of this region-specific DNA hypomethylation state, region-specific epigenomic manipulation is essential as an analytical method. We have applied genome editing to show that region-specific DNA methylation can be effectively introduced by a fusion protein, TALE, which recognizes pericentromeres, and SssI, a bacterial CpG methyltransferase. This makes it possible to increase the DNA methylation state of the pericentromeres, which is normally about 20%, to about 60-75%, enabling comparative analysis of the developmental processes of normal embryos with hypomethylated pericentromeres and embryos that have been epigenetically edited to be hypermethylated. In this chapter, we describe a method for introducing DNA methylation into pericentromeres of early mouse embryos by expressing TALE-SssI fusion protein and a method for detecting DNA methylation.

Androgen-regulated MafB drives cell migration via MMP11-dependent extracellular matrix remodeling in mice.

While androgen is considered a pivotal regulator of sexually dimorphic development, it remains unclear how it orchestrates the differentiation of reproductive organs. Using external genitalia development as a model, we showed that androgen, through the transcription factor MafB, induced cell migration by remodeling the local extracellular matrix (ECM), leading to increased cell contractility and focal adhesion assembly. Furthermore, we identified the matrix metalloproteinase Mmp11 as a MafB target gene under androgen signaling. MMP11 remodels the local ECM environment by degrading Collagen VI (ColVI). The reduction of ColVI led to the fibrillar deposition of fibronectin in the MafB-expressing bilateral mesenchyme both in vivo and ex vivo. The ECM remodeling and development of migratory cell characteristics were lost in the MafB loss-of-function mice. These results demonstrate the requirement of mesenchymal-derived androgen signaling on ECM-dependent cell migration, providing insights into the regulatory cellular mechanisms underlying androgen-driven sexual differentiation.

Sex-specific Association of Primary Aldosteronism With Visceral Adiposity.

Context: The association between primary aldosteronism and obesity, especially its sex difference, remains unknown. Objective: To assess the association for each subtype of primary aldosteronism with obesity parameters including visceral adipose tissue and differences between sexes. Methods: In this case-control study, 4 normotensive controls were selected for each case with primary aldosteronism. Multivariable conditional logistic regression models were used to estimate the association between each type of primary aldosteronism and obesity indicators. We used a random forest to identify which visceral or subcutaneous tissue areas had a closer association with disease status. Results: The study subjects included 42 aldosterone-producing adenoma cases (22 women) and 68 idiopathic hyperaldosteronism cases (42 women). In multivariable conditional logistic regressions, aldosterone-producing adenoma was significantly associated with body mass index only in men (odds ratio [OR] [95% CI)], 4.62 [1.98-10.80] per 2.89 kg/m2) but not in women (OR [95% CI], 1.09 [0.69-1.72] per 3.93 kg/m2) compared with the matched controls, whereas idiopathic hyperaldosteronism was associated with body mass index in both men (OR [95% CI], 3.96 [2.03-7.73] per 3.75 kg/m2) and women (OR [95% CI], 2.65 [1.77-3.96] per 3.85 kg/m2) compared with the matched controls. In random forests, visceral adipose tissue areas were the better predictor of both aldosterone-producing adenoma and idiopathic hyperaldosteronism than subcutaneous adipose tissue. Conclusions: Aldosterone-producing adenoma cases were obese among men, but not among women. Idiopathic hyperaldosteronism cases were obese among both men and women. Visceral adipose tissue may contribute to the pathophysiology of primary aldosteronism.

Asynchronous division at 4-8-cell stage of preimplantation embryos affects live birth through ICM/TE differentiation.

To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4-8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.

DNA replication fork speed underlies cell fate changes and promotes reprogramming.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.

Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging.

In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the 40 chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Association of achieved blood pressure after treatment for primary aldosteronism with long-term kidney function.

Little is known regarding the association of blood pressure (BP) after treatment for primary aldosteronism (PA) (i.e., adrenalectomy and mineralocorticoid receptor antagonists) with long-term renal outcomes, and whether the association is independent of BP before treatment. Using a dataset from a nationwide registry of PA in Japan, we assessed whether achieved BP levels 6 months after treatment for PA are associated with annual changes in estimated glomerular filtration rate (eGFR), rapid eGFR decline, and incident chronic kidney disease (CKD) during the 5-year follow-up period. The cohort included 1266 PA patients. In multivariable linear regression including systolic BP (SBP) levels before treatment for PA, estimates (95% confidence interval [CI]) for annual changes in eGFR after month 6 associated with one-standard deviation (1-SD) higher SBP at month 6 were -0.08 (-0.15, -0.02) mL/min/1.73 m2/year. After multivariable adjustment, the estimate (95% CI) for annual changes in eGFR after month 6 was -0.12 (-0.21, -0.02) for SBP ≥ 130 mmHg vs. SBP < 130 mmHg at month 6. Among 537 participants without CKD at baseline, a 1-SD higher SBP was associated with a higher risk for incident CKD events (hazard ratio [95% CI]: 1.40 [1.00, 1.94]). Higher SBP after treatment for PA was associated with a higher risk for kidney dysfunction over time, independently of BP levels before treatment. Achieving SBP lower than 130 mmHg after treatment for PA may be linked to better kidney outcomes.

Anatomical-functional discordance between quantitative coronary angiography and diastolic pressure ratio during wave-free period.

OBJECTIVES: This study sought to determine the predictors of anatomical-functional discordance between quantitative coronary angiography (QCA) derived diameter stenosis (QCA-DS) and diastolic pressure ratio during wave-free period (dPRWFP ). BACKGROUND: The discrepancy between angiographical stenosis and physiological significance is frequently experienced in clinical practice. Although the anatomical-functional discordance between angiography and fractional flow reserve (FFR) has been intensively investigated, that of resting index including dPRWFP remains to be elucidated. METHODS: In a total of 647 angiographically intermediate lesions with QCA-DS between 30 and 70% in 502 patients, predictors of having QCA-DS >50% and dPRWFP > 0.89 (QCA-dPRWFP mismatch), and those of having QCA-DS ≤50% and dPRWFP ≤ 0.89 (QCA-dPRWFP reverse mismatch) were determined. FFR ≤0.80 was defined as positive FFR and the predictors of QCA-FFR discordance were determined as well. RESULTS: QCA-dPRWFP mismatch and reverse mismatch were observed in 27.5 and 17.6% of cases, respectively. The predictors of mismatch were non-left anterior descending artery (LAD) lesion, large minimal lumen diameter, low baseline heart rate, and high coronary flow reserve (CFR), while those of reverse mismatch were LAD lesion, non-culprit lesion of acute coronary syndrome, long lesion length, low left ventricular ejection fraction, and low CFR and index of microcirculatory resistance. Age, sex, and the culprit vessel of prior myocardial infarction were not significant determinants of QCA-dPRWFP discordance unlike QCA-FFR discordance derived from the same cohort. CONCLUSIONS: Anatomical-functional discordance between angiography and dPRWFP was not uncommon. Predictors differed between QCA-dPRWFP discordance and QCA-FFR discordance.

Erratum: Yamazaki et al. Editing DNA Methylation in Mammalian Embryos. Int. J. Mol. Sci. 2020, 21, 637.

The authors wish to make the following corrections to our previously published paper [...].

The Population-Attributable Fraction for Premature Mortality Due to Cardiovascular Disease Associated With Stage 1 and 2 Hypertension Among Japanese.

BACKGROUND: Our aim was to assess how the population-attributable fraction (PAF) for premature mortality due to cardiovascular disease (CVD) associated with hypertension changes if blood pressure (BP) thresholds for hypertension were lowered from systolic/diastolic BP ≥140/90 mm Hg to ≥130/80 mm Hg, as defined using the 2017 American College of Cardiology/American Heart Association blood pressure guideline. METHODS: Analyses were conducted using a database of participants who underwent a national health checkup examination started in 2008 in Japan (n = 510,238; mean age, 59.6 ± 8.1 years; 42% men). Each participant was categorized as having normal or elevated BP, or stage 1 or 2 hypertension according to the guideline. Data on premature mortality due to CVD occurring before age 70 years were available through March 2015. RESULTS: Over a median follow-up of 3.4 years, 739 deaths from CVD occurred. After multivariable adjustment, hazard ratios for premature CVD mortality for elevated BP, stage 1 hypertension, and stage 2 hypertension vs. normal BP were 1.02 (95% confidence interval, 0.72, 1.44), 1.33 (1.02, 1.75), and 2.41 (1.90, 3.05), respectively. The PAF associated with stage 1 and 2 hypertension was 4.4% and 39.4%, respectively. CONCLUSIONS: In the current nationwide study of Japanese adults, stage 1 and 2 hypertension were associated with an increased risk for premature CVD mortality. The PAF for premature CVD mortality associated with hypertension increased by 4.4% if BP thresholds for hypertension were lowered from systolic/diastolic BP ≥140/90 to ≥130/80 mm Hg.

Predictive variables for hemodialysis and death in Japanese spotted fever, and the association between distance from rivers and incidence.

The number of patients with Japanese spotted fever (JSF) and its case fatality rate have been increasing in Japan and other East Asian countries. Better clinical and laboratory biomarkers are needed to avoid misdiagnosing JSF and to predict severe cases. In addition to determining these predictors, we aimed to examine the association between the incidence of JSF and the distance from rivers, in Hiroshima Prefecture, one of the most JSF prevalent areas in Japan. Patients diagnosed with JSF from 2009 to 2017 in two hospitals in Onomichi City in Hiroshima Prefecture were studied, and their clinical characteristics and laboratory data were collected retrospectively from medical charts. A random forest was used to identify predictors of severe JSF leading to hemodialysis or death. A multivariable negative binomial regression model was utilized to analyze the association between the cumulative incidence in each postal code area and the distance from the residential postal code area to the closest river. Out of 82 patients with JSF (mean age at diagnosis, 74.1 ± 10.6 years; 34 (41.5 %) men), 6 cases were regarded as severe (among them 5 hemodialysis patients and 3 deaths). Twenty-eight (34.1 %) patients were misdiagnosed at least once at the initial hospital visit. Laboratory examination showed 34.5 % had atypical lymphocytes, 73.8 % had no eosinophils, 75.6 % had an elevated aspartate aminotransferase (AST) level, and 69.5 % had hyponatremia. Among cases without urine leucocytes, 63.3 % had proteinuria and 63.3 % had hematuria. Low serum total protein was the strongest predictor of severe JSF, followed by high blood urea nitrogen (BUN) and low albumin. Geospatial analysis showed a significant negative association between the cumulative incidence of JSF cases and the distance from rivers in an adjusted model: the cumulative incidence decreased by 0.51 times (95 % CI: 0.30 to 0.86) for every kilometer of distance from the residential postal code area to the closest river. Some laboratory data may be useful in averting misdiagnosis of JSF and in predicting severe cases. Additional studies should be done in order to clarify the mechanism and association of the incidence of JSF with the distance from the nearest river.

Galactosialidosis Type IIb with Bilateral Macular Cherry-Red Spots but Mild Dysfunction.

Galactosialidosis is a rare metabolic disorder resulting from mutations in the CTSA gene. Few studies have reported on the ocular findings of galactosialidosis type IIb in detail. We report on a case of galactosialidosis, the diagnosis of which was suggested by bilateral macular cherry-red spots, which is an indication of lysosomal storage disease. In this case, retinal and systemic dysfunctions were mild. Genetic studies revealed an abnormality of relevant protective proteins, and thus a definitive diagnosis was made. The patient was a 35-year-old man who had blurred vision from young age, but he did not seek any therapy due to good visual acuity. He visited a local clinic after the blurred vision in the left eye worsened and was referred to us for bilateral macular cherry-red spots. He had no family history of note. We observed fine grayish-white deposits in the corneal stroma and fine opacity of the lens. Optical coherence tomography showed a hyperreflective region and a thick bilateral retinal ganglion cell layer. Goldmann perimetry showed focal loss of sensitivity. There was almost no functional decline noted on multifocal electroretinography. Lysosomal storage disease was suspected due to corneal clouding and macular cherry-red spots, and so further evaluation was performed. Though neurological abnormality was mild, we made a diagnosis of galactosialidosis because of decreased activity of ß-galactosidase and sialidase. Genetic studies revealed an abnormality of relevant protective proteins. Since the onset was later in life and clinical symptoms were mild, we expect that the ophthalmological findings will remain stable. Long-term observation is necessary for this case.

Ostial left circumflex lesion with calcified nodule successfully treated with excimer laser coronary atherectomy and drug-coated balloon.

Stenting for severely calcified lesions has a higher risk of stent restenosis or stent failure than stenting for lesions without calcification, and stenting for complex lesions including ostial or bifurcation lesions sometimes causes plaque shift which leads to side branch occlusion. A calcified nodule (CN) is considered one of the culprits for stable angina or acute coronary syndrome. However, the optimal strategy for this lesion is not well clarified. We report a patient who presented stable angina with a CN at the ostial left circumflex artery. In this case, pretreatment with excimer laser coronary atherectomy (ELCA) and scoring balloon dilatation followed by drug-coated balloon (DCB) dilatation successfully prevented plaque shift caused by stenting in the acute phase. In addition, it also maintained the patency in the late phase. Furthermore, we observed the CN lesions at preprocedural, postprocedural, and late phase by optical coherence tomography. ELCA, which has a unique debulking technique, and scoring balloon dilatation followed by DCB dilatation might offer an alternative treatment for ostial CN lesions instead of stenting. 〈Learning objective: The optimal strategy for severely calcified lesions with calcified nodule is controversial because the prevalence of calcified nodule is rare and stent failure is more common in calcified lesions. In particular, regarding a calcified nodule located in ostial left circumflex coronary artery lesion, excimer laser coronary atherectomy and scoring balloon dilatation followed by drug-coated balloon may give an alternative treatment to avoid stenting.〉.

Serum Alanine Transaminase as a Predictor of Type 2 Diabetes Incidence: The Yuport Prospective Cohort Study.

Prior studies have shown an association between the incidence of diabetes with liver enzymes, such as alanine transaminase (ALT). Liver fibrosis scores, such as the Fibrosis-4 index which indicates chronic liver damage, were also associated with diabetes development. However, no literature compared predictive accuracy between ALT and Fibrosis-4 index. Thus, we aimed to determine it, and to assess its association using inverse probability of treatment weighting. This was a non-concurrent prospective cohort study of 9,748 subjects without diabetes receiving Yuport Health Checkup in Japan between 1998 and 2006. ALT was categorized into three groups: the highest ALT group (men ≥ 30 U/L and women ≥ 20 U/L), the middle (men ≥ 20 and < 30 U/L, and women ≥ 14 and < 20 U/L), and the lowest (men < 20 U/L and women < 14 U/L). The primary outcome was the new onset of diabetes. The area under the receiver operating characteristic curves (AUC) of ALT for predicting the diabetes development was higher than that of any other markers of liver damage. The AUC for ALT was 0.71, while that for the Fibrosis-4 index was 0.51 (p < 0.001 for the difference between the AUCs). The highest and middle ALT groups had a significantly higher incidence of diabetes than the lowest group: adjusted relative risk 1.79 [95% confidence interval (CI): 1.29, 2.58], and 1.64 [95% CI: 1.17, 2.38] respectively. Of the various indicators of liver function, ALT is likely to be the most accurate and associated predictor of diabetes development.

Comparison of Neointimal Response between Durable-Polymer Everolimus-Eluting Stent and Bioabsorbable-Polymer Everolimus-Eluting Stent for Severely Calcified Lesions Requiring Rotational Atherectomy.

Clinical outcomes after percutaneous coronary intervention (PCI) for severely calcified lesions remain poor. The purpose of this study was to investigate the neointimal response after everolimus-eluting stents (EES) for severely calcified lesions treated with rotational atherectomy (RA) using optical coherence tomography (OCT).We retrospectively analyzed 34 lesions in which PCI was performed with EES deployment following RA and OCT was performed immediately after PCI and at follow-up (nine months). The EES was either durable-polymer (DP) EES (22 lesions) or bioabsorbable polymer (BP) -EES (12 lesions). Strut coverage and malapposition were evaluated at 1-mm intervals of cross-section (CS) by serial OCT analysis. Malapposed strut was defined as having the distance from luminal border > 100 µm.A total of 11,823 struts immediately after PCI and 11,720 struts at follow-up were analyzed. Immediately after PCI, the strut-level analysis showed no significant differences in the percentage of malapposed struts between the DP-EES group and the BP-EES group. At follow-up, the BP-EES group showed a more prevalent covered strut compared with the DP-EES group (strut-level analysis: 95% versus 97%, P = 0.045; CS-level analysis: 97% versus 100%, P < 0.01; lesion-level analysis: 27% versus 83%, P < 0.01, respectively).In severely calcified lesions requiring RA, the BP-EES group achieved better neointimal coverage than the DP-EES group at nine months. Additional prospective studies are needed.