Induction of homologous recombination by site-specific replication stress.

DNA replication stress is one of the primary causes of genome instability. In response to replication stress, cells can employ replication restart mechanisms that rely on homologous recombination to resume replication fork progression and preserve genome integrity. In this review, we provide an overview of various methods that have been developed to induce site-specific replication fork stalling or collapse in eukaryotic cells. In particular, we highlight recent studies of mechanisms of replication-associated recombination resulting from site-specific protein-DNA barriers and single-strand breaks, and we discuss the contributions of these findings to our understanding of the consequences of these forms of stress on genome stability.

Tournament Recovery Profiles and Physical Demands in a Collegiate Women's Tennis Team.

ABSTRACT: Tendero-Ortiz, E, Johnson, MJ, Horsfall, CM, Vondrasek, JD, Grosicki, GJ, Riemann, BL, and Flatt, AA. Tournament recovery profiles and physical demands in a collegiate women's tennis team. J Strength Cond Res XX(X): 000-000, 2024-We aimed to characterize recovery profiles and tournament physical demands in women's collegiate tennis players. A Division 1 team (n = 9) participated in the study. Markers of cardiac autonomic (resting heart rate [HR], HR variability), neuromuscular (isometric handgrip strength, seated single-arm shot-put test [SSAPT], hexagon agility, countermovement jump characteristics), and perceptual recovery were obtained before the tournament (baseline) and again 1 and 2 days posttournament. Cardiorespiratory (HR) and movement characteristics from matches were quantified with wearable devices. p values < 0.05 were statistically significant. No recovery markers differed from baseline (ps > 0.05), although small effect size reductions 1 day posttournament were noted for SSAPT, hexagon agility, and select countermovement jump characteristics. In addition, hexagon agility times and SSAPT were slower (p < 0.01) and shorter (p < 0.05), respectively, at 1 versus 2 days posttournament. Similarly, relative to 1 day posttournament, perceptual makers were improved 2 days posttournament (ps < 0.05). Mean and peak HR were higher for singles versus doubles matches  (ps < 0.05). Except for average speed, movement parameters were greater during singles versus doubles matches (ps < 0.05). Markers of recovery were minimally affected 1 day posttournament relative to baseline, but perceptual and select neuromuscular markers were most improved 2 days posttournament. Thus, passive rest or limited intensity training 1 day posttournament seems advisable. Competition HR and movement profiles inform practitioners of the cardiorespiratory and locomotor demands of women's collegiate tennis, which may be useful in designing preparatory conditioning programs to ensure that players attain match-specific physical capacities in training before competition.

Strand asymmetry in the repair of replication dependent double-strand breaks.

Single-strand breaks (SSBs) are one of the most common endogenous lesions and have the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate the mechanism of replication fork collapse at SSBs and subsequent repair, we employed Cas9 nickase (nCas9) to generate site and strand-specific nicks in the budding yeast genome. We show that nCas9-induced nicks are converted to mostly double-ended DSBs during S-phase. We find that repair of replication-dependent DSBs requires homologous recombination (HR) and is independent of canonical non-homologous end joining. Consistent with a strong bias to repair these lesions using a sister chromatid template, we observe minimal induction of inter-chromosomal HR by nCas9. Using nCas9 and a gRNA to nick either the leading or lagging strand template, we carried out a genome-wide screen to identify factors necessary for the repair of replication-dependent DSBs. All the core HR genes were recovered in the screen with both gRNAs, but we recovered components of the replication-coupled nucleosome assembly (RCNA) pathway with only the gRNA targeting the leading strand template. By use of additional gRNAs, we find that the RCNA pathway is especially important to repair a leading strand fork collapse.

Engineering Memory T Cells as a platform for Long-Term Enzyme Replacement Therapy in Lysosomal Storage Disorders.

Enzymopathy disorders are the result of missing or defective enzymes. Amongst these enzymopathies, mucopolysaccharidosis type I, is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), ultimately causes toxic build-up of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS). Human memory T cells (Tm) migrate throughout the body's tissues and can persist for years, making them an attractive approach for cellular-based, systemic enzyme replacement therapy. Here, we tested genetically engineered, IDUA-expressing Tm as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm take up residence in nearly all tested tissues, producing IDUA and leading to metabolic correction of GAG levels in the heart, lung, liver, spleen, kidney, bone marrow, and the CNS. Our study indicates that genetically engineered Tm holds great promise as a platform for cellular-based enzyme replacement therapy for the treatment of mucopolysaccharidosis type I and potentially many other enzymopathies and protein deficiencies.

Residual Effects of Same Day Lower Extremity Strength Training on Countermovement Jump Performance in Collegiate Women Athletes.

Balancing of strength programming intensity with sport demands is necessary to avoid excessive workloads that could inhibit performance. To expand previous jump height focused literature, this study evaluated whether countermovement jump (CMJ) movement strategies, including eccentric characteristics, might reveal CMJ execution strategy shifts to achieve similar afternoon CMJ height following a morning resistance training session (RTS). Fifteen collegiate women's soccer and volleyball athletes (18-24 years, 73.6 ± 8.4 kg, 1.74 ± 0.19 m) participating in an offseason RTS completed five CMJs during two afternoon sessions (48 h apart), one 4-6 h post morning RTS, and one on a rest day. The RTS consisted of 2 sets of 10 repetitions at 70-80% 1RM for the back squat, the front squat, and the forward lunge. Vertical ground reaction forces were recorded from which 13 outcome measures describing elements of the eccentric and concentric CMJ phases were computed. No significant differences in jump height (p = 0.427, d = 0.17) or outcome measures (p = 0.091-0.777, d = -0.07-0.21) between sessions with exception of a significant concentric phase time decrease (p = 0.026, d = 0.23) following the RTS were identified. Given the magnitude of the mean concentric phase time change (0.01 s), the result likely has limited practical meaning. As these results confirm previous CMJ height literature, practitioners have further evidence that a morning RTS does not interfere or enhance afternoon CMJ performance in athletic women.

Outcomes of open reduction and internal fixation of calcaneus fractures: A database study comparing patients with and without diabetes.

Treatment of calcaneal fractures in patients with diabetes mellitus (DM) is challenging. The purpose of this study was to compare post-operative outcomes after open reduction and internal fixation (ORIF) for calcaneus fracture in patients with complicated DM, uncomplicated DM, and patients without DM. A commercially available de-identified database was queried for all calcaneus fracture diagnoses undergoing ORIF from 2010 to 2021. The patients were separated into three groups for analysis: patients without DM (10,951, 82.6%), uncomplicated DM (1,500, 11.3%) and complicated DM (802, 6.1%). At 1 year, post-operative adverse events were assessed among the three groups. The odds of adverse event(s) for each group were compared between the three groups with and without characteristic matching. In the unmatched cohorts, patients with complicated DM, when compared with patients without DM and patients with uncomplicated DM, had significantly higher rates of all adverse events with exception of DVT. Rates of CNA were significantly higher in patients with complicated DM compared with no DM (OR 107.7 (CI 24.83-467.6) p < 0.0001) and uncomplicated DM (OR 44.26 (CI 3.86-507.93) p = 0.0002). After matching, non-union, AKI, sepsis, surgical site infection, and wound disruption were higher in patients with complicated DM compared with patients without DM. There were no significant differences in the three groups with regard to reoperation, DVT, MI, pneumonia, or below the knee amputation. Patients with DM who underwent ORIF for calcaneus fracture experienced higher rates of post-operative adverse events compared with those patients without DM.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Fractures and dislocations of the foot and ankle in people with diabetes: a literature review.

Diabetes (DM) increases fracture risk, and bone quality depends on type diabetes type, duration, and other comorbidities. Diabetes is associated with a 32% increased relative risk (RR) of total fractures and 24% increased RR of ankle fractures compared with patients without DM. Type 2 DM is associated with a 37% increased RR of foot fractures compared with patients without DM. The incidence of ankle fractures in the general population is 169/100,000 per year, while foot fractures occur less frequently, with an incidence of 142/100,000 per year. Biomechanical properties of bone are negatively impacted by stiff collagen, contributing to the increased risk of fragility fractures in patients with DM. Systemic elevation of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), interleukin-1ß (IL-1ß), and interleukin 6 (IL-6), impact bone healing in patients with DM. Fractures in patients with DM, can be associated with poorly regulated levels of RANKL (receptor activator of nuclear transcription factor kappa-b ligand) leading to prolonged osteoclastogenesis, and net bone resorption. One of the most salient factors in treating fractures and dislocations of the foot and ankle is to recognize the difference between patients with uncomplicated and complicated DM. Complicated diabetes is defined as 'end organ damage', and for the purposes of this review, includes patients with neuropathy, peripheral artery disease (PAD) and/or chronic renal disease. Uncomplicated diabetes is not associated with 'end organ damage'. Foot and ankle fractures in patients with complicated DM pose challenges, and surgery is associated with increased risks of impaired wound healing, delayed fracture healing, malunion, infection, surgical site infection, and revision surgery. While patients with uncomplicated DM can be treated like patients without DM, patients with complicated DM require close follow-up and robust fixation methods should be considered to withstand the anticipated prolonged healing period. The aims of this review are as follows: (1) to review pertinent aspects of DM bone physiology and fracture healing, (2) to review the recent literature on treatment of foot and ankle fractures in patients with complicated DM, and (3) to provide treatment protocols based on the recent published evidence.

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation.

Homologous recombination (HR), the high-fidelity mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5'-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely linked repeats, but are required for interchromosomal repeat recombination in Saccharomyces cerevisiae. This context-specific requirement for long-range end resection is connected to its role in activating the DNA damage checkpoint. Consistent with this role, checkpoint mutants also show a defect specifically in interchromosomal recombination. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1∆ sgs1∆ cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1∆ sgs1∆ cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.

Ankle Joint Salvage for Rigid Flatfoot Deformity.

Rigid flatfoot deformity with valgus ankle instability is a complex condition to treat. Thorough clinical and radiographic evaluation is vital to determine treatment strategies. Nonoperative treatment usually relies on bracing or various orthoses. Surgical interventions include ligament reconstruction, osteotomies, arthrodesis, arthroplasty, or a combination of these procedures. Before addressing the ankle deformity, a plantigrade foot is important so a staged approach may be necessary. Misalignment of the ankle replacement can lead to edge loading and early failure. As the implants and our understanding of ankle arthroplasty improve, more patients may benefit from a motion-preserving procedure rather than an arthrodesis.

A Stepwise Approach to Nonoperative and Operative Management of the Diabetic Foot Ulceration.

There are multiple factors that lead to the development of the diabetic foot ulceration (DFU). The ultimate goal when treating DFU is to prevent amputation. Initial therapy should include debridement, maintaining a healthy wound environment, and offloading. In the setting of infection or a nonhealing DFU, surgical intervention may be necessary. The goals of this article are to discuss the key aspects of the initial examination, standard nonoperative treatment, and the operative treatment options for patients with DFU.

Limb Salvage in Severe Diabetic Foot Infection.

Severe diabetic foot infections (DFI) are both limb threatening and life threatening and associated with negative impact on health-related quality of life. Most severe DFIs require surgical intervention, and the goal of treatment should be preservation of limb function in addition to eradication of infection. Minor amputations are required in approximately 40% and major amputations in approximately 20% of patients. Significant risk factors for lower extremity amputation included male gender, smoking, previous amputation, osteomyelitis, peripheral artery disease, retinopathy, severe infections, gangrene, neuroischemic diabetic foot infections, leukocytosis, positive wound cultures, and isolation of gram-negative bacteria.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade.

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

A Systematic Review of Intramedullary Fixation in Midfoot Charcot Neuroarthropathy.

Charcot neuroarthropathy can cause severe deformity of the midfoot, and intramedullary use of beams and bolts has been utilized as a method of definitive stabilization. This systematic review evaluated the outcomes of intramedullary beaming in patients with Charcot neuroarthropathy and determined the methodological quality of the studies. Four online databases were searched: PubMed, MEDLINE (Clarivate Analytics), CINAHL (Cumulative Index to Nursing and Allied Health) and Web of Science (Clarivate Analytics). To assess the methodological quality of the studies, the Coleman Methodology Score was used. The data was pooled into 2 outcomes groups for comparison: (1) Studies that reported on the outcomes of Charcot specific implants (study group). (2) Studies that reported on the outcomes using non-Charcot specific implants (control group). After screening, 16 studies were included. Compared to our control group, our study group had significantly higher rates of overall hardware complications, hardware migration, surgical site infection, reoperation, and nonunion. The study group had significantly lower rates of limb salvage compared to the control group. Our study and control groups did not differ in the rates of hardware breakage, wound healing complications, or mortality. The limb salvage rate was 92% and 97% of patients were still alive at a mean follow-up of 25 months. The mean Coleman Methodology Score indicated the quality of the studies was poor and consistent with methodologic limitations. The quality of published studies on intramedullary implants for Charcot reconstruction is low. Complications when utilizing intramedullary fixation for Charcot reconstruction are high, whether or not Charcot specific implants are used.

Amputation and infection are the greatest fears in patients with diabetes foot complications.

AIMS: To determine the degree patients with diabetic foot ulcers, Charcot neuroarthropathy and neuropathic fractures and dislocations fear complications (death, dialysis, heart attack, stroke, blindness, diabetic foot infection, minor and major lower extremity amputation [LEA]) that can occur and to assess if there is a difference between fears of patients with diabetic foot ulcers, Charcot neuroarthropathy and neuropathic fractures and dislocations and diabetic patients without these complications. METHODS: 478 patients completed an eight question Likert scale survey. The study group was defined as non-infected foot ulcers, neuropathic fractures and Charcot neuroarthropathy. RESULTS: Of the 478 patients, 121 (25.3 %) had diabetic foot ulcers, Charcot neuroarthropathy or neuropathic fractures and dislocations and 357 (74.7 %) did not. The study group had significantly higher odds of reporting extreme fear of foot infection (OR 2.8, 95 % CI 1.8-4.5), major LEA (OR 2.8, 95 % CI 1.8-4.4), minor LEA (OR 2.3, 95 % CI 1.5-3.5), blindness (OR 2.0, 95 % CI 1.3-3.2), dialysis (OR 2.0, 95 % CI 1.1-3.3), and death (OR 2.4, 95 % CI 1.4-4.2). In the study group highest rated fear measures were foot infection (3.71, SD 1.23), minor amputation (3.67, SD 1.45) and major amputation (3.63, SD 1.52). There were no significant differences in the mean fear of infection, minor amputation or major amputation. CONCLUSION: Patients with diabetic foot ulcers, Charcot neuroarthropathy or neuropathic fractures and dislocations reported higher fear ratings of diabetes-related complications compared to those without these complications.

Arthroscopic Findings in Refractory Symptomatic Fourth and Fifth Tarsometatarsal Joints.

Refractory pain to the fourth and fifth tarsometatarsal (TMT) joint can be a source of disability and functional impairment. While pain has been attributed to injury, post-traumatic arthritis, arthrofibrosis, the principal causes of pain in the absence of arthritis are not well elucidated. The purpose of this study is to characterize arthroscopic pathology associated with chronic refractory pain to the fourth and fifth TMT joints. We retrospectively examined 24 patients that underwent arthroscopic surgery of the fourth and fifth TMT joints for refractory pain at our academic institution between 2015 and 2019. We used the Outerbridge classification for chondral lesions, the Kellgren Lawrence radiographic classification for osteoarthritis, and described intraarticular pathologies as acute hypertrophic synovitis, chronic synovial fibrosis, hyaline bands, meniscoid bodies, loose joint bodies, arthrofibrosis. Approximately, 31 of 45 TMT joints (68.9%) presented with radiographic evidence of arthritis. Approximately, 14 of 45 TMT joints (31.11%) were absent of radiographic signs of arthritis. The frequency of soft tissue pathology seen in these patients without radiographic evidence of arthritis was arthrofibrosis (87.5%), chronic synovial fibrosis (75.0%), and acute hypertrophic synovitis (62.5%). This is the first study to report arthroscopic pathologies associated with refractory pain to the fourth and fifth TMT joints.

Identifying Risk Factors for Nonunion of the Modified Lapidus Procedure for the Correction of Hallux Valgus.

There is a paucity of literature characterizing risk factors for nonunion associated with the modified Lapidus procedure for correction of hallux valgus. The purpose of this study was to evaluate risk factors associated with nonunion for Lapidus bunionectomies. Patients who underwent modified Lapidus procedure from 2009 to 2018 were retrospectively reviewed. Patient's age, sex, body mass index, prior bunionectomy, history of tobacco use, presence of diabetes mellitus or hypothyroidism, and fixation method were recorded along with pre- and postoperative radiographic parameters. A multiple logistic regression analysis was implemented to estimate the odds of nonunion. Of the 222 patients who met inclusion criteria, nonunion with modified Lapidus procedure was observed in 20 patients (9.01%). Odds of nonunion with modified Lapidus procedure were greater for patients who had undergone previous bunionectomy (odds ratio [OR] = 3.957, 95% confidence interval [CI]: 1.021-15.338), as body mass index increased (OR = 1.091, 95% CI: 1.018-1.170), and as preoperative HV angle increased (OR = 1.108, 95% CI: 1.020-1.203). Odds of nonunion were lower for patients as preoperative intermetatarsal angle increased (OR = 0.739, 95% CI: 0.580-0.941). No significant increased odds of nonunion were found between fixation methods.

Body Composition and Cardiometabolic Risk Markers in Children of Women who Took Part in a Randomized Controlled Trial of a Preconceptional Nutritional Intervention in Mumbai, India.

BACKGROUND: Maternal nutrition influences fetal development and may permanently alter ("program") offspring body composition and metabolism, thereby influencing later risk of diabetes and cardiovascular (cardiometabolic) disease. The prevalence of cardiometabolic disease is rising rapidly in India. OBJECTIVES: To test the hypothesis that supplementing low-income Indian women with micronutrient-rich foods preconceptionally and during pregnancy has a beneficial impact on the children's body composition and cardiometabolic risk marker profiles. METHODS: Follow-up of 1255 children aged 5-10 y whose mothers took part in the Mumbai Maternal Nutrition Project [Project "SARAS"; International Standard Randomised Controlled Trial Number (ISRCTN)62811278]. Mothers were randomly assigned to receive a daily micronutrient-rich snack or a control snack of lower micronutrient content, both made from local foods, in addition to normal diet, from before pregnancy until delivery. Children's body composition was assessed using anthropometry and DXA. Their blood pressure, plasma glucose, insulin, and lipid concentrations were measured. Outcomes were compared between allocation groups with and without adjustment for confounding factors. RESULTS: Overall, 15% of children were stunted, 34% were wasted, and 3% were overweight. In the intention-to-treat analysis, there were no differences in body composition or risk markers between children in the intervention and control groups. Among children whose mothers started supplementation ≥3 mo before conception (the "per protocol" sample) the intervention increased adiposity among girls, but not boys. BMI in girls was increased relative to controls by 2% (95% CI: 1, 4; P = 0.01); fat mass index by 10% (95% CI: 3, 18; P = 0.004); and percent fat by 7% (95% CI: 1, 13; P = 0.01) unadjusted, with similar results in adjusted models. CONCLUSIONS: Overall, supplementing women with micronutrient-rich foods from before pregnancy until delivery did not alter body composition or cardiometabolic risk markers in the children. Subgroup analyses showed that, if started ≥3 mo before conception, supplementation may increase adiposity among female children.

Body Composition and Cardiometabolic Risk Markers in Children of Women who Took Part in a Randomized Controlled Trial of a Preconceptional Nutritional Intervention in Mumbai, India.

BACKGROUND: Maternal nutrition influences fetal development and may permanently alter ("program") offspring body composition and metabolism, thereby influencing later risk of diabetes and cardiovascular (cardiometabolic) disease. The prevalence of cardiometabolic disease is rising rapidly in India. OBJECTIVES: To test the hypothesis that supplementing low-income Indian women with micronutrient-rich foods preconceptionally and during pregnancy has a beneficial impact on the children's body composition and cardiometabolic risk marker profiles. METHODS: Follow-up of 1255 children aged 5-10 y whose mothers took part in the Mumbai Maternal Nutrition Project [Project "SARAS"; International Standard Randomised Controlled Trial Number (ISRCTN)62811278]. Mothers were randomly assigned to receive a daily micronutrient-rich snack or a control snack of lower micronutrient content, both made from local foods, in addition to normal diet, from before pregnancy until delivery. Children's body composition was assessed using anthropometry and DXA. Their blood pressure, plasma glucose, insulin, and lipid concentrations were measured. Outcomes were compared between allocation groups with and without adjustment for confounding factors. RESULTS: Overall, 15% of children were stunted, 34% were wasted, and 3% were overweight. In the intention-to-treat analysis, there were no differences in body composition or risk markers between children in the intervention and control groups. Among children whose mothers started supplementation ≥3 mo before conception (the "per protocol" sample) the intervention increased adiposity among girls, but not boys. BMI in girls was increased relative to controls by 2% (95% CI: 1, 4; P = 0.01); fat mass index by 10% (95% CI: 3, 18; P = 0.004); and percent fat by 7% (95% CI: 1, 13; P = 0.01) unadjusted, with similar results in adjusted models. CONCLUSIONS: Overall, supplementing women with micronutrient-rich foods from before pregnancy until delivery did not alter body composition or cardiometabolic risk markers in the children. Subgroup analyses showed that, if started ≥3 mo before conception, supplementation may increase adiposity among female children.