Gene Expression Profiling in Pediatric Appendicitis.

Importance: Appendicitis is the most common indication for urgent surgery in the pediatric population, presenting across a range of severity and with variable complications. Differentiating simple appendicitis (SA) and perforated appendicitis (PA) on presentation may help direct further diagnostic workup and appropriate therapy selection, including antibiotic choice and timing of surgery. Objective: To provide a mechanistic understanding of the differences in disease severity of appendicitis with the objective of developing improved diagnostics and treatments, specifically for the pediatric population. Design, Setting, and Participants: The Gene Expression Profiling of Pediatric Appendicitis (GEPPA) study was a single-center prospective exploratory diagnostic study with transcriptomic profiling of peripheral blood collected from a cohort of children aged 5 to 17 years with abdominal pain and suspected appendicitis between November 2016 and April 2017 at the Alberta Children's Hospital in Calgary, Alberta, Canada, with data analysis reported in August 2023. There was no patient follow-up in this study. Exposure: SA, PA, or nonappendicitis abdominal pain. Main Outcomes and Measures: Blood transcriptomics was used to develop a hypothesis of underlying mechanistic differences between SA and PA to build mechanistic hypotheses and blood-based diagnostics. Results: Seventy-one children (mean [SD] age, 11.8 [3.0] years; 48 [67.6%] male) presenting to the emergency department with abdominal pain and suspected appendicitis were investigated using whole-blood transcriptomics. A central role for immune system pathways was revealed in PA, including a dampening of major innate interferon responses. Gene expression changes in patients with PA were consistent with downregulation of immune response and inflammation pathways and shared similarities with gene expression signatures derived from patients with sepsis, including the most severe sepsis endotypes. Despite the challenges in identifying early biomarkers of severe appendicitis, a 4-gene signature that was predictive of PA compared to SA, with an accuracy of 85.7% (95% CI, 72.8-94.1) was identified. Conclusions: This study found that PA was complicated by a dysregulated immune response. This finding should inform improved diagnostics of severity, early management strategies, and prevention of further postsurgical complications.

The Dictyostelium discoideum FimA protein, unlike yeast and plant fimbrins, is regulated by calcium similar to mammalian plastins.

Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory 'switch helix' that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.

Clinical and metabolomic characterization of Brivanib-Induced hypertension in metastatic colorectal cancer.

BACKGROUND: Trials of tyrosine kinase inhibitors (TKI) have not demonstrated dramatic benefits in advanced colorectal cancer (CRC), and this may be a function of poor patient selection. TKI-induced hypertension is reportedly a surrogate marker for treatment benefit for some tumor types. Our objective was to determine whether hypertension was associated with benefit in the context of CRC treatment, and also to gain insight on the pathogenesis of TKI-induced hypertension by monitoring associated changes in the circulating metabolome. PATIENTS AND METHODS: Clinical data were acquired from clinical trial patients with metastatic CRC randomized to cetuximab ± the TKI brivanib (N = 750). Outcomes were evaluated as a function of treatment-induced hypertension. For metabolomic studies, plasma samples were taken at baseline, as well as at 1, 4, and 12 weeks after treatment initiation. Samples were submitted to gas chromatography-mass spectrometry to identify treatment-related metabolomic changes associated with TKI-induced hypertension, compared to pre-treatment baseline. A model based on changes in metabolite concentrations was generated using orthogonal partial least squares discriminant analysis (OPLS-DA). RESULTS: In the brivanib treated group, 95 patients had treatment-related hypertension within 12 weeks of initiating treatment. TKI-induced hypertension was not associated with a significantly higher response rate, nor was it associated with improved progression-free or overall survival. In metabolomic studies, 386 metabolites were identified. There were 29 metabolites that changed with treatment and distinguished patients with and without TKI-induced hypertension. The OPLS-DA model for brivanib-induced hypertension was significant and robust (R2 Y score = 0.89, Q2 Y score = 0.70, CV-ANOVA = 2.01 e-7). Notable metabolomic features previously reported in pre-eclampsia and associated with vasoconstriction were found. CONCLUSION: TKI-induced hypertension was not associated with clinical benefit in metastatic CRC. We have identified changes in the metabolome that are associated with the development of worsening brivanib-induced hypertension that may be useful in future efforts of characterizing this toxicity.

A combined computational-biophysical approach to understanding fatty acid binding to FABP7.

Members of the fatty acid binding protein (FABP) family function as intracellular transporters of long-chain fatty acids and other hydrophobic molecules to different cellular compartments. Brain FABP (FABP7) exhibits ligand-directed differences in cellular transport. For example, when FABP7 binds to docosahexaenoic acid (DHA), the complex relocates to the nucleus and influences transcriptional activity, whereas FABP7 bound with monosaturated fatty acids remains in the cytosol. Preferential binding of FABP7 to polyunsaturated fatty acids like DHA has been previously observed and is thought to play a role in differential localization. However, we find that at 37°C, FABP7 does not display strong selectivity, suggesting that the conformational ensemble of FABP7 and its perturbation upon binding may be important. We use molecular dynamics simulations, NMR, and a variety of biophysical techniques to better understand the conformational ensemble of FABP7, how it is perturbed by fatty acid binding, and how this may be related to ligand-directed transport. We find that FABP7 has high degree of conformational heterogeneity that is substantially reduced upon ligand binding. We also observe substantial heterogeneity in ligand binding poses, which is consistent with our finding that ligand binding is resistant to mutations in key polar residues in the binding pocket. Our NMR experiments show that DHA binding leads to chemical shift perturbations in residues near the nuclear localization signal, which may point toward a mechanism of differential transport.

Metabolomics in pediatric lower respiratory tract infections and sepsis: a literature review.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality in children. The ability of healthcare providers to diagnose and prognose LRTIs in the pediatric population remains a challenge, as children can present with similar clinical features regardless of the underlying pathogen or ultimate severity. Metabolomics, the large-scale analysis of metabolites and metabolic pathways offers new tools and insights that may aid in diagnosing and predicting the outcomes of LRTIs in children. This review highlights the latest literature on the clinical utility of metabolomics in providing care for children with bronchiolitis, pneumonia, COVID-19, and sepsis. IMPACT: This article summarizes current metabolomics approaches to diagnosing and predicting the course of pediatric lower respiratory infections. This article highlights the limitations to current metabolomics research and highlights future directions for the field.

Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues.

Cecropin P1 (CP1) isolated from a large roundworm Ascaris suum, which is found in pig intestines, has been extensively studied as a model antimicrobial peptide (AMP). However, despite being a model AMP, its antibacterial mechanism is not well understood, particularly the function of its C-terminus. By using an Escherichia coli overexpression system with calmodulin as a fusion partner, we succeeded in the mass expression of recombinant peptides, avoiding toxicity to the host and degradation of CP1. The structure of the recombinant 15N- and 13C-labeled CP1 and its C-terminus truncated analogue in dodecylphosphocholine (DPC) micelles was determined by NMR. In this membrane-mimetic environment, CP1 formed an α-helix for almost its entire length, except for a short region at the C-terminus, and there was no evidence of a hinge, which is considered important for the expression of activity in other cecropins. Several NMR analyses showed that the entire length of CP1 was protected from water by micelles. Since the loss of the C-terminus of the analogue had little effect on the NMR structure or its interaction with the micelle, we investigated another role of the C-terminus of CP1 in its antimicrobial activity. The results showed that the C-terminal region affected the DNA-binding capacity of CP1, and this mechanism of action was also newly suggested that it contributed to the antimicrobial activity of CP1.

Comparison of Two Automated Targeted Metabolomics Programs to Manual Profiling by an Experienced Spectroscopist for 1H-NMR Spectra.

Automated programs that carry out targeted metabolite identification and quantification using proton nuclear magnetic resonance spectra can overcome time and cost barriers that limit metabolomics use. However, their performance needs to be comparable to that of an experienced spectroscopist. A previously analyzed pediatric sepsis data set of serum samples was used to compare results generated by the automated programs rDolphin and BATMAN with the results obtained by manual profiling for 58 identified metabolites. Metabolites were selected using Student's t-tests and evaluated with several performance metrics. The manual profiling results had the highest performance metrics values, especially for sensitivity (76.9%), area under the receiver operating characteristic curve (0.90), precision (62.5%), and testing accuracy based on a neural net (88.6%). All three approaches had high specificity values (77.7-86.7%). Manual profiling by an expert spectroscopist outperformed two open-source automated programs, indicating that further development is needed to achieve acceptable performance levels.

Early detection of treatment futility in patients with metastatic colorectal cancer.

PURPOSE: Chemotherapy options for treating CRC have rapidly expanded in recent years, and few have predictive biomarkers. Oncologists are challenged with evidence-based selection of treatments, and response is evaluated retrospectively based on serial imaging beginning after 2-3 months. As a result, cumulative toxicities may appear in patients who will not benefit. Early recognition of non-benefit would reduce cumulative toxicities. Our objective was to determine treatment-related changes in the circulating metabolome corresponding to treatment futility. METHODS: Metabolomic studies were performed on serial plasma samples from patients with CRC in a randomized controlled trial of cetuximab vs. cetuximab + brivanib (N = 188). GC-MS quantified named 94 metabolites and concentrations were evaluated at baseline, Weeks 1, 4 and 12 after treatment initiation. In a discovery cohort (N = 68), a model distinguishing changes in metabolites associated with radiographic disease progression and response was generated using OPLS-DA. A cohort of 120 patients was used for validation of the model. RESULTS: By one week after treatment, a stable model of 21 metabolites could distinguish between progression and partial response (R2Y = 0.859; Q2Y = 0.605; P = 5e-4). In the validation cohort, patients with the biomarker had a significantly shorter OS (P < 0.0001). In a separate cohort of patients with HCC on axitinib, appearance of the biomarker also signified a shorter PFS (1.7 months vs. 9.2 months, P = 0.001). CONCLUSION: We have identified changes in the metabolome that appear within 1 week of starting treatment associated with treatment futility. The novel approach described is applicable to future efforts in developing a biomarker for early assessment of treatment efficacy.

Simultaneous binding of the N- and C-terminal cytoplasmic domains of aquaporin 4 to calmodulin.

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 µM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; Kd1: 3.6 µM, Kd2: 113.6 µM, respectively). A recombinant AQP4 protein domain (recAQP4CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 µM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data support a unique adapter protein binding mode for CaM with AQP4.

Maternal Metabolites Indicative of Mental Health Status during Pregnancy.

Approximately 25% of individuals report poor mental health during their pregnancy or postpartum period, which may impact fetal neurodevelopment, birth outcomes, and maternal behaviors. In the present study, maternal serum samples were collected from pregnancies at 28-32 weeks gestation from the All Our Families (Alberta, Canada) cohort and assessed using nuclear magnetic resonance spectroscopy (1H-NMR) and inductively coupled plasma-mass spectrometry (ICP-MS). Individuals with poor mental health at 34-36 weeks gestation were age-matched with mentally healthy pregnant controls. Metabolites were examined against validated self-reported mental health questionnaires for associations with depressive symptoms (Edinburgh Perinatal Depression Scale) and anxiety symptoms (Spielberger State-Trait Anxiety Inventory). 1H-NMR metabolites were identified for depression (alanine, leucine, valine, methionine, phenylalanine, glucose, lactate, 3-hydroxybutyrate, and pyruvate) and anxiety (3-hydroxybutyrate). For ICP-MS, antimony and zinc were significant for depression and anxiety, respectively. Upon false discovery rate (FDR) correction at 10%, five 1H-NMR metabolites (alanine, leucine, lactate, glucose, and phenylalanine) for depression remained significantly increased. Although results warrant further validation, the identified metabolites may serve as a predictive tool for assessing mental health during pregnancy as earlier identification has the potential to aid intervention and management of poor mental health symptomology, thus avoiding harmful consequences to both mother and offspring.

Metabolomics and Inflammatory Mediator Profiling for the Differentiation of Life-Threatening and Non-Severe Appendicitis in the Pediatric Population.

While children with appendicitis often have excellent clinical outcomes, some develop life-threatening complications including sepsis and organ dysfunction requiring pediatric intensive care unit (PICU) support. Our study applied a metabolomics and inflammatory protein mediator (IPM) profiling approach to determine the bio-profiles of children who developed severe appendicitis compared with those that did not. We performed a prospective case-control study of children aged 0-17 years with a diagnosis of appendicitis. Cases had severe disease resulting in PICU admission. Primary controls had moderate appendicitis (perforation without PICU); secondary controls had mild appendicitis (non-perforated). Serum samples were analyzed using Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy and Gas Chromatography-Mass Spectrometry (GC-MS); IPM analysis was performed using plasma bead-based multiplex profiling. Comparisons were made using multivariate data statistical analysis. Fifty-three children were included (15 severe, 38 non-severe). Separation between severe and moderate appendicitis demonstrated excellent sensitivity and specificity (100%, 88%; 14 compounds), separation between severe and mild appendicitis also showed excellent sensitivity and specificity (91%, 90%; 16 compounds). Biomarker patterns derived from metabolomics and IPM profiling are capable of distinguishing children with severe appendicitis from those with less severe disease. These findings provide an important first step towards developing non-invasive diagnostic tools for clinicians in early identification of children who are at a high risk of developing severe appendicitis.

An Integrative Approach to Determine 3D Protein Structures Using Sparse Paramagnetic NMR Data and Physical Modeling.

Paramagnetic nuclear magnetic resonance (NMR) methods have emerged as powerful tools for structure determination of large, sparsely protonated proteins. However traditional applications face several challenges, including a need for large datasets to offset the sparsity of restraints, the difficulty in accounting for the conformational heterogeneity of the spin-label, and noisy experimental data. Here we propose an integrative approach to structure determination combining sparse paramagnetic NMR with physical modelling to infer approximate protein structural ensembles. We use calmodulin in complex with the smooth muscle myosin light chain kinase peptide as a model system. Despite acquiring data from samples labeled only at the backbone amide positions, we are able to produce an ensemble with an average RMSD of ∼2.8 Å from a reference X-ray crystal structure. Our approach requires only backbone chemical shifts and measurements of the paramagnetic relaxation enhancement and residual dipolar couplings that can be obtained from sparsely labeled samples.

Seminal plasma metabolomics reveals lysine and serine dysregulation as unique features distinguishing between prostate cancer tumors of Gleason grades 6 and 7.

BACKGROUND: Prostate cancer (PCa) is a metabolic disease. Most men are diagnosed with low grade indolent disease and differentiating these men from those who have life threatening cancer is a challenging but important clinical dilemma. There are currently limited biomarkers that can distinguish between the indolent Gleason grade 6 and higher-grade disease. Moreover, some individuals initially diagnosed with low grade disease progress to higher grade disease. Currently prostate biopsies are the only reliable methods of stratifying risk, but biopsies can cause significant morbidity, sample only a small portion of the gland and are costly. Therefore, biomarkers distinguishing between indolent and aggressive patterns of PCa are urgently required to minimize biopsy-associated morbidity, prevent over-treatment of indolent PCa and to better stratify patients for appropriate treatment. METHODS: Seminal fluid samples were collected from normal individuals (n = 13) Before infertility treatment and histologically confirmed PCa patients (n = 51). 1 H Nuclear magnetic resonance spectroscopy and orthogonal partial least square discriminant analysis were used to compare the populations. RESULTS: Alterations in amino acids levels, specifically lysine and serine and changes in glycolytic intermediates were the most significant metabolic features associated with differences between healthy controls and PCa and between Gleason grade 6 (GS6) and Gleason grade 7 (GS7) samples. Orthogonal partial least square plots discriminated healthy controls from PCa samples (R 2 = 0.54, Q 2 = 0.31; area under the receiver operating characteristics curve [AUC] = 0.96), and GS6 from GS7 samples (R 2 = 0.62, Q 2 = 0.49; AUC = 0.98) based on lysine and serine content. CONCLUSION: This study suggests that seminal plasma metabolomics profiling of seminal fluid is a promising means of differentiating indolent from aggressive disease. Particularly, lysine and serine levels may be able to differentiate GS6 from GS7 disease.

ARDS metabolic fingerprints: characterization, benchmarking, and potential mechanistic interpretation.

In this study, we aimed to identify acute respiratory distress syndrome (ARDS) metabolic fingerprints in selected patient cohorts and compare the metabolic profiles of direct versus indirect ARDS and hypoinflammatory versus hyperinflammatory ARDS. We hypothesized that the biological and inflammatory processes in ARDS would manifest as unique metabolomic fingerprints that set ARDS apart from other intensive care unit (ICU) conditions and could help examine ARDS subphenotypes and clinical subgroups. Patients with ARDS (n = 108) and ICU ventilated controls (n = 27) were included. Samples were randomly divided into 2/3 training and 1/3 test sets. Samples were analyzed using 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Twelve proteins/cytokines were also measured. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to select the most differentiating ARDS metabolites and protein/cytokines. Predictive performance of OPLS-DA models was measured in the test set. Temporal changes of metabolites were examined as patients progressed through ARDS until clinical recovery. Metabolic profiles of direct versus indirect ARDS subgroups and hypoinflammatory versus hyperinflammatory ARDS subgroups were compared. Serum metabolomics and proteins/cytokines had similar area under receiver operator curves when distinguishing ARDS from ICU controls. Pathway analysis of ARDS differentiating metabolites identified a dominant involvement of serine-glycine metabolism. In longitudinal tracking, the identified pathway metabolites generally exhibited correction by 7-14 days, coinciding with clinical improvement. ARDS subphenotypes and clinical subgroups were metabolically distinct. However, our identified metabolic fingerprints are not ARDS diagnostic biomarkers, and further research is required to ascertain generalizability. In conclusion, patients with ARDS are metabolically different from ICU controls. ARDS subphenotypes and clinical subgroups are metabolically distinct.

Harnessing the Benefits of Neuroinflammation: Generation of Macrophages/Microglia with Prominent Remyelinating Properties.

Excessive inflammation within the CNS is injurious, but an immune response is also required for regeneration. Macrophages and microglia adopt different properties depending on their microenvironment, and exposure to IL4 and IL13 has been used to elicit repair. Unexpectedly, while LPS-exposed macrophages and microglia killed neural cells in culture, the addition of LPS to IL4/IL13-treated macrophages and microglia profoundly elevated IL10, repair metabolites, heparin binding epidermal growth factor trophic factor, antioxidants, and matrix-remodeling proteases. In C57BL/6 female mice, the generation of M(LPS/IL4/IL13) macrophages required TLR4 and MyD88 signaling, downstream activation of phosphatidylinositol-3 kinase/mTOR and MAP kinases, and convergence on phospho-CREB, STAT6, and NFE2. Following mouse spinal cord demyelination, local LPS/IL4/IL13 deposition markedly increased lesional phagocytic macrophages/microglia, lactate and heparin binding epidermal growth factor, matrix remodeling, oligodendrogenesis, and remyelination. Our data show that a prominent reparative state of macrophages/microglia is generated by the unexpected integration of pro- and anti-inflammatory activation cues. The results have translational potential, as the LPS/IL4/IL13 mixture could be locally applied to a focal CNS injury to enhance neural regeneration and recovery.SIGNIFICANCE STATEMENT The combination of LPS and regulatory IL4 and IL13 signaling in macrophages and microglia produces a previously unknown and particularly reparative phenotype devoid of pro-inflammatory neurotoxic features. The local administration of LPS/IL4/IL13 into spinal cord lesion elicits profound oligodendrogenesis and remyelination. The careful use of LPS and IL4/IL13 mixture could harness the known benefits of neuroinflammation to enable repair in neurologic insults.

Multimodal peripheral fluid biomarker analysis in clinically isolated syndrome and early multiple sclerosis.

BACKGROUND: Increasing evidence suggests that various inflammatory, immunological and metabolic pathways are altered in the clinically isolated syndrome (CIS) of multiple sclerosis (MS). Moreover, recent diagnostic criteria have made possible the very early diagnosis of MS. We evaluated multiple fluid biomarkers in people with early MS and CIS. METHODS: We measured blood levels of cytokines, matrix metalloproteinases (MMPs), serum metabolomics and immune cell immunophenotyping in participants in the Trial of Minocycline in a Clinically Isolated Syndrome of Multiple Sclerosis. RESULTS: When compared with healthy controls, people with early MS/CIS had higher levels of eotaxin, MCP-3, IL-1 receptor antagonist, IL-1ß, IL-9 and IP-10, as well as MMPs 1, 8 and 9. In metabolomics analysis, the alanine, aspartate and glutamate metabolism and the synthesis and degradation of ketone bodies pathways were altered compared to healthy controls. There were no differences in lymphocyte subpopulation numbers. Out of all these biomarkers, only MMP-1 was able to differentiate between early MS and CIS, and was found to correlate with lesion volume and gadolinium enhancing lesions on MRI. CONCLUSION: The immunological and metabolic profile of CIS and early MS is remarkably similar, supporting that these are a continuum of a common underlying pathophysiological process.

Maternal and Cord Blood Metabolite Associations with Gestational Weight Gain and Pregnancy Health Outcomes.

Pre-pregnancy obesity and excessive gestational weight gain (GWG) are risk factors for future maternal and childhood obesity. Maternal obesity is potentially communicated to the fetus in part by the metabolome, altering the child's metabolic program in early development. Fasting maternal blood samples from 37 singleton pregnancies at 25-28 weeks of gestation were obtained from mothers with pre-pregnancy body mass indexes (BMIs) between 18 and 40 kg/m2. Various health measures including GWG, diet, and physical activity were also assessed. At term (37-42 weeks), a venous umbilical cord sample was obtained. Serum metabolomic profiles were measured using nuclear magnetic resonance spectroscopy as well as a gut and metabolic hormone panel. Maternal and cord serum metabolites were tested for associations with pre-pregnancy BMI, GWG, health outcomes, and gut and metabolic hormones. While cord blood metabolites showed no significant correlation to maternal obesity status or other measured health outcomes, maternal serum metabolites showed distinct profiles for lean, overweight, and obese women. Additionally, four serum metabolites, namely, glutamate, lysine, pyruvate, and valine, allowed prediction of excessive GWG when pre-pregnancy BMI was controlled. Metabolic biomarkers predictive of GWG are reported and, if validated, could aid in the guidance of prenatal weight management plans as the majority of pregnancy weight gain occurs in the third trimester.