Tear fluid cytokine analysis: a non-invasive approach for assessing retinopathy of prematurity severity.

PURPOSE: To determine whether there is a significant association between inflammatory cytokines in the tear fluid and the severity of Retinopathy of Prematurity (ROP). STUDY DESIGN: Retrospective cohort study. METHODS: The cytokine levels in tear fluids were determined in 34 eyes with ROP and 18 eyes without ROP. There were 15 eyes with severe ROP requiring treatment and 19 eyes with mild ROP not requiring treatment. For severe ROP eyes, tear fluids were collected before treatment. RESULTS: Significantly higher levels of CCL2 and vascular endothelial growth factor (VEGF) were detected in eyes with severe ROP compared to eyes with mild ROP and no ROP. When assessed for cytokine levels that discriminate each disease group, CCL2 showed a significant odds ratio of 1.76 for severity change (/quintile, P = 0.032, after adjusting for birth weight). Correlation analysis showed that birth weight correlated with IL-1α levels, and decreased weight gain increased IFN-γ levels. We next determined tear fluid cytokines which discriminate severe ROP using receiver operating characteristics' analysis. We found that combination of higher CCL2 levels, higher VEGF levels, and lower IFN-γ levels in the tear fluid had a stronger predictive value for severe ROP (area under curve, 0.85). CONCLUSION: The levels of CCL2, VEGF, and IFN-γ in tear fluid may serve as useful biomarkers for assessing the severity of ROP.

Use of amlodipine in the treatment of cats with systemic hypertension in Japan.

An increase in systemic blood pressure causes bleeding and ischemia owing to peripheral vascular breakdown, leading to various forms of organ damage. The brain, eyes, kidneys, and cardiovascular system are known target organs for hypertension. To our knowledge, no reports in Japan describe, in detail, the types of antihypertensive drugs used to treat hypertension in cats or its underlying causes. Therefore, we aimed to investigate the use of antihypertensive drugs in domestic cats with hypertension in Japan, the causes of hypertension, and the vital prognosis of these patients. In the present survey, we found that amlodipine was used alone (60/80 cats) or concomitantly (20/80 cats) in all cat patients with hypertension in Japan. We also determined that blood pressure measurements were not yet routinely performed on cats at veterinary clinics in Japan. Furthermore, we have new information suggesting that amlodipine administration in cats with hypertension, which lowers systolic arterial pressure levels to within the normal range (<140 mmHg), may have a negative impact on their survival. Routine blood pressure measurements for cats during their regular health checkups can help identify hypertension, and proper interpretation of blood pressure readings can facilitate suitable treatment measures.

Development of sexual dimorphism of skeletal muscles through the adrenal cortex, caused by androgen-induced global gene suppression.

The zona fasciculata (zF) in the adrenal cortex contributes to multiple physiological actions through glucocorticoid synthesis. The size, proliferation, and glucocorticoid synthesis characteristics are all female biased, and sexual dimorphism is established by androgen. In this study, transcriptomes were obtained to unveil the sex differentiation mechanism. Interestingly, both the amount of mRNA and the expressions of nearly all genes were higher in females. The expression of Nr5a1, which is essential for steroidogenic cell differentiation, was also female biased. Whole-genome studies demonstrated that NR5A1 regulates nearly all gene expression directly or indirectly. This suggests that androgen-induced global gene suppression is potentially mediated by NR5A1. Using Nr5a1 heterozygous mice, whose adrenal cortex is smaller than the wild type, we demonstrated that the size of skeletal muscles is possibly regulated by glucocorticoid synthesized by zF. Taken together, considering the ubiquitous presence of glucocorticoid receptors, our findings provide a pathway for sex differentiation through glucocorticoid synthesis.

Profiling miRNAs in tear extracellular vesicles: a pilot study with implications for diagnosis of ocular diseases.

PURPOSE: To estimate the roles of extracellular vesicles (EVs) in tears and to determine whether their profiles are associated with the type of ocular disease. STUDY DESIGN: Cross-sectional study. METHODS: Tear EVs were extracted from 14 healthy participants and from 21 patients with retinal diseases (age-related macular degeneration [AMD] or diabetic macular edema [DME]). The surface marker expression of tear EVs was examined, and microRNAs (miRNAs) were extracted and profiled by use of real-time PCR array. The stability of the expression of the miRNAs was determined, and their functions were assessed by network analyses. Classification accuracy was evaluated by use of a random forest classifier and k-fold cross-validation. RESULTS: The miRNAs that were highly expressed in tear EVs were miR-323-3p, miR-548a-3p, and miR-516a-5p. The most stably expressed miRNAs independent of diseases were miR-520h and miR-146b-3p. The primary networks of the highly stably expressed endogenous miRNAs were annotated as regulation of organismal injury and abnormalities. The highly expressed miRNAs for severe retinal disease were miR-151-5p for AMD and miR-422a for DME, suggesting potential roles of tear EVs in liquid biopsy. Nine miRNAs (miR-25, miR-30d, miR-125b, miR-132, miR-150, miR-184, miR-342-3p, miR-378, and miR-518b) were identified as distinguishing individuals with AMD from healthy individuals with a classification accuracy of 91.9%. CONCLUSIONS: The finding that tear EVs contain characteristic miRNA species indicates that they may help in maintaining homeostasis and serve as a potential tool for disease diagnosis.

Increased infiltration of CD4+ T cell in the complement deficient lymphedema model.

BACKGROUND: Lymphedema is an intractable disease that can be caused by injury to lymphatic vessels, such as by surgical treatments for cancer. It can lead to impaired joint mobility in the extremities and reduced quality of life. Chronic inflammation due to infiltration of various immune cells in an area of lymphedema is thought to lead to local fibrosis, but the molecular pathogenesis of lymphedema remains unclear. Development of effective therapies requires elucidation of the immunological mechanisms involved in the progression of lymphedema. The complement system is part of the innate immune system which has a central role in the elimination of invading microbes and acts as a scavenger of altered host cells, such as apoptotic and necrotic cells and cellular debris. Complement-targeted therapies have recently been clinically applied to various diseases caused by complement overactivation. In this context, we aimed to determine whether complement activation is involved in the development of lymphedema. RESULTS: Our mouse tail lymphedema models showed increased expression of C3, and that the classical or lectin pathway was locally activated. Complement activation was suggested to be involved in the progression of lymphedema. In comparison of the C3 knockout (KO) mouse lymphedema model and wild-type mice, there was no difference in the degree of edema at three weeks postoperatively, but the C3 KO mice had a significant increase of TUNEL+ necrotic cells and CD4+ T cells. Infiltration of macrophages and granulocytes was not significantly elevated in C3 KO or C5 KO mice compared with in wild-type mice. Impaired opsonization and decreased migration of macrophages and granulocytes due to C3 deficiency should therefore induce the accumulation of dead cells and may lead to increased infiltration of CD4+ T cells. CONCLUSIONS: Vigilance for exacerbation of lymphedema is necessary when surgical treatments have the potential to injure lymphatic vessels in patients undergoing complement-targeted therapies or with complement deficiency. Future studies should aim to elucidate the molecular mechanism of CD4+ T cell infiltration by accumulated dead cells.

A cryptic root isolate belonging to Geoglossales from potted Rhododendron: its molecular phylogeny and ability to colonize an ericoid mycorrhizal host in vitro.

Although the lifestyle of Geoglossales remains largely unknown, recent advancements have established a hypothesis regarding the ericoid mycorrhizal lifestyle of geoglossoid fungi. In this study, we focused on one isolate of Geoglossales sp. obtained from surface-sterilized roots of potted Rhododendron transiens. We aimed to reveal the phylogenetic position and in vitro colonizing ability of this species in the hair roots of ericoid mycorrhizal plants. Based on our multigene phylogenetic tree, this species is a sister of the genus Sarcoleotia which has not been reported from either other studies or field environment. Its ascocarps could not be obtained, and conspecific sequences were not found in the databases and repositories examined. The Geoglossales sp. colonized the vital rhizodermal cells of blueberries in vitro with hyphal coils. There were relatively large morphological variations of coils consistent with extraradical hyphae; however, overall, the colonization morphologically resembled those by Sarcoleotia globosa and representative ericoid mycorrhizal fungi. The taxonomy and ecological significance of the species remain to be resolved; nevertheless, our results suggest that the ericoid mycorrhizal lifestyle may be widespread within Geoglossales.

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry.

We report the first sequencing of morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomers, PMOs) using electron capture dissociation (ECD) mass spectrometry. In this research, we found dissociation of the backbone of 18- to 25-mer PMOs to produce d and z ions as the major ions, and 100% cleavage coverage (sequence coverage) was obtained with these ions. This is a critical contrast with beam-type collision-induced dissociation, which dominantly induces base loss, so it is difficult to obtain sequence information. The results showed that an electron beam energy (typically 15 eV) can be used universally for PMOs with different sequences, lengths, and charge states so that no detailed optimization is required for multiprecursor targeting liquid chromatography coupled with tandem mass spectrometry measurements. We also confirmed that the ECD reaction speed was compatible with the high-performance liquid chromatography time scale. Finally, we demonstrated a liquid chromatography electron capture dissociation tandem mass spectrometry workflow to survey the modification sites of the emulated PMO impurities.

Analyses of Dupilumab-Related Ocular Adverse Drug Reactions Using the WHO's VigiBase.

INTRODUCTION: Dupilumab is a drug that inhibits the action of interleukin (IL)-4 and IL-13 and is a potent therapeutic drug for allergic diseases such as atopic dermatitis. Although its use has been associated with significant ocular adverse drug reactions (ADRs), the IL-4 and IL-13 inhibition may also have favorable therapeutic effects. The aim of this study was to determine the disease spectrum in which the use of dupilumab may have been associated with an increase or decrease of ocular ADRs. METHODS: We searched the World Health Organization's VigiBase for ADRs associated with the use of dupilumab for data up to 12 June 2022. The number of all ADRs that were retrieved was compared with the number of ocular ADRs associated with the use of dupilumab. Disproportionate reporting was assessed by calculating the information component (IC) values and odds ratios. RESULTS: Since the introduction of dupilumab, 100,267 ADRs have been reported. Of all the ADRs associated with dupilumab, 28,522 ADRs were ocular complications, and it ranked fourth in the ocular complications by organ level. By assessments of the IC for age ≤ 44 years, the most significantly associated ADRs were dry eye followed by blepharitis including eyelid crusting and dryness and conjunctivitis. Crusting and dryness of the eyelids were the most significant ADRs for all age groups. Other ocular ADRs reported include meibomian gland dysfunction, keratitis, glaucoma, and retinal disorders. In contrast, periorbital edema, neuro-ophthalmic disorders, optic neuritis, and macular edema were significantly reduced by the use of dupilumab. CONCLUSIONS: Dupilumab-related ADRs included an increase or decrease of various ocular disorders. The results indicate that dupilumab also has potential therapeutic effects.

Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography-Electron-Activated Dissociation Time-of-Flight Mass Spectrometry.

Herein, we report a novel liquid chromatography coupled with tandem mass spectrometry method to characterize N-acetylneuraminic acid (Neu5Ac, Sa) linkage in N-linked glycans in glycopeptides with no sialic acid derivatization. First, we established a separation in reversed-phase high-performance liquid chromatography (HPLC) using a higher formic acid concentration in the mobile phases, which separated the N-glycopeptides depending on the Sa linkage. We also demonstrated a novel characterization method of Sa linkages in N-glycopeptides using electron-activated dissociation. We found that hot electron capture dissociation using an electron beam energy higher than 5 eV cleaved glycosidic bonds in glycopeptides, resulting in each glycosidic bond in the antennas being broken on both sides of the oxygen atom. Such glycosidic bond cleavage at the reducing end (C-type ion) showed the difference in Sa linkages between Sa-Gal, Gal-GlcNAc, and GlcNAc-Man. We proposed a rule to characterize the Sa linkages using the Sa-Gal products. This method was applied to N-glycopeptides in tryptic fetuin digest separated by an optimized reversed-phase HPLC. We successfully identified a number of isomeric glycoforms in the glycopeptides with different Sa links, whose peptide backbones were also simultaneously sequenced by hot ECD.

LPGAT1/LPLAT7 regulates acyl chain profiles at the sn-1 position of phospholipids in murine skeletal muscles.

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.

Immediate Effect of an Intervention Using a Head-mounted Display with a Modified Pitch Angle on Spatial Awareness and Standing Balance in Stroke Hemiparesis.

Objectives: This study aimed to clarify the effect of an intervention using a head-mounted display with a web camera set at a modified pitch angle on spatial awareness, sit-to-stand movement, and standing balance in patients with left and right hemisphere damage. Methods: The participants were 12 patients with right hemisphere damage and 12 patients with left hemisphere damage. The line bisection test, a sit-to-stand movement, and balance assessment were performed before and after the intervention. The intervention task involved pointing at targets 48 times in an upward bias condition. Results: Significant upward deviation on the line bisection test was noted in patients with right hemisphere damage. The load on the forefoot during the sit-to-stand movement was significantly increased. The range of anterior-posterior sway during forward movement in the balance assessment was reduced. Conclusions: An adaptation task performed in an upward bias condition may produce an immediate effect on upward localization, sit-to-stand movement, and balance performance in patients with right hemisphere stroke.

Association of IL-4 with pachychoroid neovasculopathy.

The purpose of this study was to identify the inflammatory cytokines that were associated with pachychoroid neovasculopathy (PNV). Seventy-five eyes of 75 patients with PNV, 145 eyes of 145 patients with neovascular age-related macular degeneration without pachyvessels, and 150 eyes of 150 normal subjects were examined for the levels of intraocular cytokines. In eyes with PNV, the levels of IL-1α, IL-1ß, IL-2, IL-4, IL-10, and VEGF were significantly higher than that of the controls. Logistic regression analysis showed that the highest association with the pachyvessels was found for IL-4, IL-2, and IL-1α. In eyes with PNV, the levels of IL-4, IL-2, IL-5, IL-13, IL-1α, and IL-1ß were significantly higher in eyes with both increased choroidal thickness and choroidal vessel diameter. The strongest correlation with the choroidal thickness and vessel diameter was observed for IL-4. In PNV eyes with polypoidal lesions, the levels of IL-4, IL-17, and TNFß were significantly correlated with the number of polypoidal lesions. Of these cytokines, IL-4 was especially associated with the thickness of the choroidal vessels and the formation of polypoidal lesions. We conclude that IL-4 is most likely involved in establishing the clinical characteristics of PNV and polypoidal vascular remodeling.

Structural Identification of Eicosanoids with Ring Structures Using Differential Mobility Spectrometry-Electron Impact Excitation of Ions from Organics Mass Spectrometry.

We developed a structural identification method for eicosanoids with various ring structures using mass spectrometry. We discovered that an electron beam with a kinetic energy of 10 eV, which is in the Electron Impact Excitation of Ions from Organics (EIEIO) regime, cleaved the fatty acids enough to distinguish constitutional and cis/trans isomers. In addition to EIEIO, a comparison to authentic standards using differential mobility spectrometry (DMS) can identify diastereomers, which was difficult by EIEIO. The combination of EIEIO and DMS can provide a high-throughput method to identify complete structures of eicosanoids in mixed samples, which is not allowed with conventional analytical methods though eicosanoids are important signaling molecules in biosystems.

Top-Down Characterization and Intact Mass Quantitation of a Monoclonal Antibody Drug from Serum by Use of a Quadrupole TOF MS System Equipped with Electron-Activated Dissociation.

Time-of-flight MS systems for biopharmaceutical and protein characterization applications may play an even more pivotal role in the future as biotherapeutics increase in drug pipelines and as top-down MS approaches increase in use. Here, a recently developed TOF MS system is examined for monoclonal antibody (mAb) characterization from serum samples. After immunocapture, purified drug material spiked into monkey serum or dosed for an in-life study is analyzed by top-down MS. While characterization aspects are a distinct advantage of the MS platform, MS system and software capabilities are also shown regarding intact protein quantitation. Such applications are demonstrated to help enable comprehensive protein molecule quantitation and characterization by use of TOF MS instrumentation.

Prostaglandin E2 receptor Ptger4b regulates female-specific peptidergic neurons and female sexual receptivity in medaka.

In vertebrates, female receptivity to male courtship is highly dependent on ovarian secretion of estrogens and prostaglandins. We recently identified female-specific neurons in the medaka (Oryzias latipes) preoptic area that express Npba, a neuropeptide mediating female sexual receptivity, in response to ovarian estrogens. Here we show by transcriptomic analysis that these neurons express a multitude of neuropeptides, in addition to Npba, in an ovarian-dependent manner, and we thus termed them female-specific, sex steroid-responsive peptidergic (FeSP) neurons. Our results further revealed that FeSP neurons express a prostaglandin E2 receptor gene, ptger4b, in an ovarian estrogen-dependent manner. Behavioral and physiological examination of ptger4b-deficient female medaka found that they exhibit increased sexual receptivity while retaining normal ovarian function and that their FeSP neurons have reduced firing activity and impaired neuropeptide release. Collectively, this work provides evidence that prostaglandin E2/Ptger4b signaling mediates the estrogenic regulation of FeSP neuron activity and female sexual receptivity.

Fast Electron Detachment Dissociation of Oligonucleotides in Electron-Nitrogen Plasma Stored in Magneto Radio-Frequency Ion Traps.

We report "plasma" electron detachment dissociation (EDD), a novel electron-activated dissociation (EAD) method for the fast sequencing of oligonucleotides with a high sequence coverage. To reduce the repulsive Coulombic force between the deprotonated oligonucleotides and the electron beam, we performed EDD in a neutral electron-nitrogen (N2+) plasma stored in a magneto radio-frequency ion trap. We confirmed that plasma EDD accomplished a high sequence coverage (100%) of RNA with 40 mers in the reaction time of 10 ms using the electron beam kinetic energy of 35 eV. This new technique was applied to various modifications in oligonucleotide therapeutics (ONTs). Phosphorothioate (PS) positions showed an extremely high dissociation efficiency, i.e., 100 times higher than the standard phosphate (PO) in DNA. Locked nucleotides did not show intensive dissociation in EDD; however, collision-induced dissociation (CID) helped sequence these portions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a ZenoTOF mass spectrometer equipped with the plasma EDD technique successfully identified impurities in degraded samples.

Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway.

Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigate this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggest that thymosin ß10 (Tmsb10) is transiently upregulated in the progenitors. While studying the function of Tmsb10, we reveal that platelet-derived growth factor (PDGF) regulates ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promotes desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induces their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveils the molecular process of FLC differentiation and shows that it is cooperatively induced by DHH and PDGF.

Localization of Multiple O-Linked Glycans Exhibited in Isomeric Glycopeptides by Hot Electron Capture Dissociation.

We describe a method to obtain a comprehensive profile of multiple glycosylations in glycopeptide isoforms. We detected a wide range of abundances of various O-glycoforms in isomeric glycopeptides using hot electron capture dissociation (hot ECD) in liquid chromatography-tandem mass spectrometry. To capture low abundant glycosylated species, a prototype of a ZenoTOF 7600 system incorporating an efficient electron-activated dissociation device to perform hot ECD was operated in targeted or scheduled high-resolution multiple reaction monitoring workflows. In addition, Zeno trap pulsing was activated to enhance the sensitivity of the time-of-flight mass spectrometer. Sixty-nine O-glycopeptides of the long O-glycopeptides in tryptic bovine fetuin digest were obtained with a relative abundance range from 100 to 0.2%, which included sialylated glycans with Neu5Ac and Neu5Gc.

Electron Impact Excitation of Ions from Organics on Singly Protonated Peptides with and without Post-Translational Modifications.

We report on the dissociation of singly protonated peptides by electrons using electron-activated dissociation (EAD), which comprises electron impact excitation of ions from organics (EIEIO), electronic-excitation dissociation (EED), and electron ionization dissociation (EIoD). Various singly protonated peptides were dissociated using a recently reported fast EAD device. The dissociation can be induced through two pathways: (i) vibrational dissociation similar to collision-activated dissociation (CAD, or collision-induced dissociation, CID) by relaxation from a molecular electronic excited state to high vibrational states; and (ii) radical-induced dissociation where molecular electronic excitation is followed by homolytic cleavage. EAD is complementary to CAD as additional molecular information can be obtained; e.g., fragile PTM moieties, such as glycosylation and sulfation, can be localized. Simultaneously, the energetic production of radical z• fragments enables Leu and Ile discrimination, like in a hot ECD process. Using the fast EAD device, LC-EIEIO-time-of-flight mass spectrometry was applied to a tryptic monoclonal antibody digest containing short singly protonated peptides.

Intrauterine Hyponutrition Reduces Fetal Testosterone Production and Postnatal Sperm Count in the Mouse.

Background: Although intrauterine hyponutrition is regarded as a risk factor for the development of "testicular dysgenesis syndrome" (TDS) in the human, underlying mechanism(s) remain largely unknown. Methods: To clarify the underlying mechanism(s), we fed vaginal plug-positive C57BL/6N female mice with regular food ad libitum throughout the pregnant course (control females) (C-females) or with 50% of the mean daily intake of the C-females from 6.5 dpc (calorie-restricted females) (R-females), and compared male reproductive findings between 17.5-dpc-old male mice delivered from C-females (C-fetuses) and those delivered from R-females (R-fetuses) and between 6-week-old male mice born to C-females (C-offspring) and those born to R-females (R-offspring). Results: Compared with the C-fetuses, the R-fetuses had (1) morphologically normal external genitalia with significantly reduced anogenital distance index, (2) normal numbers of testicular component cells, and (3) significantly low intratesticular testosterone, in association with significantly reduced expressions of steroidogenic genes. Furthermore, compared with the C-offspring, the R-offspring had (1) significantly increased TUNEL-positive cells and normal numbers of other testicular component cells, (2) normal intratesticular testosterone, in association with normal expressions of steroidogenic genes, (3) significantly reduced sperm count, and normal testis weight and sperm motility, and (4) significantly altered expressions of oxidation stress-related, apoptosis-related, and spermatogenesis-related genes. Conclusions: The results, together with the previous data including the association between testosterone deprivation and oxidative stress-evoked apoptotic activation, imply that reduced fetal testosterone production is the primary underlying factor for the development of TDS in intrauterine hyponutrition, and that TDS is included in the clinical spectrum of Developmental Origins of Health and Disease.