iPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain1. Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues2-6. The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development7-10. However, current approaches do not incorporate microglia or address their role in organoid maturation11-21. Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac)22. In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis.

Vaccination and associated factors among tribal children of 1 year age in nine Indian districts: A cross-sectional study.

OBJECTIVE: To report on vaccination status by 12 months of age among tribal children from nine districts of India. METHODS: Cross-sectional study of 2631 tribal women having a child aged 12 months or below from nine Indian districts with a considerable proportion of the tribal population. Socio-demographic details, reception of various vaccines by 12 months of age, mother's antenatal care utilisation and health system-related details were collected through a pre-tested, interviewer-administered questionnaire from mothers. Multiple logistic regression analysis was used to identify the factors associated with complete vaccination by 12 months of age. RESULTS: Only 52% of children were fully vaccinated by the age of 12 months among the tribal populations; 11% did not receive any vaccine, and 37% of the tribal children received some vaccines. The age-appropriate vaccination was unsatisfactory as only 75% of the infants received all birth dose vaccines, and only 60.5% received all doses by 14 weeks. Only 73% were vaccinated against measles. Illness of the child, home births and communication gaps concerning vaccination were the main reasons for an infant not being vaccinated appropriately. Frequency of health worker's visits to the village, hospital birth, reception of advice on vaccination and educational status of the head of the households were significantly associated with full vaccination status. CONCLUSION: A relatively low proportion of children were fully vaccinated among the tribal populations. Health systems factors, mainly the outreach services and advice by the health workers, were positively and significantly associated with a child being fully vaccinated by 12 months of age. Improving outreach services is crucial to improve vaccination coverage in tribal areas, and there is a need to address the social determinants in the long run.

Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2.

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

Maintaining human milk bank services throughout the COVID-19 pandemic: A global response.

If maternal milk is unavailable, the World Health Organization recommends that the first alternative should be pasteurised donor human milk (DHM). Human milk banks (HMBs) screen and recruit milk donors, and DHM principally feeds very low birth weight babies, reducing the risk of complications and supporting maternal breastfeeding where used alongside optimal lactation support. The COVID-19 pandemic has presented a range of challenges to HMBs worldwide. This study aimed to understand the impacts of the pandemic on HMB services and develop initial guidance regarding risk limitation. A Virtual Collaborative Network (VCN) comprising over 80 HMB leaders from 36 countries was formed in March 2020 and included academics and nongovernmental organisations. Individual milk banks, national networks and regional associations submitted data regarding the number of HMBs, volume of DHM produced and number of recipients in each global region. Estimates were calculated in the context of missing or incomplete data. Through open-ended questioning, the experiences of milk banks from each country in the first 2 months of the pandemic were collected and major themes identified. According to data collected from 446 individual HMBs, more than 800,000 infants receive DHM worldwide each year. Seven pandemic-related specific vulnerabilities to service provision were identified, including sufficient donors, prescreening disruption, DHM availability, logistics, communication, safe handling and contingency planning, which were highly context-dependent. The VCN now plans a formal consensus approach to the optimal response of HMBs to new pathogens using crowdsourced data, enabling the benchmarking of future strategies to support DHM access and neonatal health in future emergencies.

A modified implementation of tristate inverter based static master-slave flip-flop with improved power-delay-area product.

The paper introduces novel architectures for implementation of fully static master-slave flip-flops for low power, high performance, and high density. Based on the proposed structure, traditional C(2)MOS latch (tristate inverter/clocked inverter) based flip-flop is implemented with fewer transistors. The modified C(2)MOS based flip-flop designs mC(2)MOSff1 and mC(2)MOSff2 are realized using only sixteen transistors each while the number of clocked transistors is also reduced in case of mC(2)MOSff1. Postlayout simulations indicate that mC(2)MOSff1 flip-flop shows 12.4% improvement in PDAP (power-delay-area product) when compared with transmission gate flip-flop (TGFF) at 16X capacitive load which is considered to be the best design alternative among the conventional master-slave flip-flops. To validate the correct behaviour of the proposed design, an eight bit asynchronous counter is designed to layout level. LVS and parasitic extraction were carried out on Calibre, whereas layouts were implemented using IC station (Mentor Graphics). HSPICE simulations were used to characterize the transient response of the flip-flop designs in a 180 nm/1.8 V CMOS technology. Simulations were also performed at 130 nm, 90 nm, and 65 nm to reveal the scalability of both the designs at modern process nodes.

Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of MgB is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

Indian Academy of Pediatrics Revised Guidelines on Evaluation, Prevention and Management of Childhood Obesity.

JUSTIFICATION: The last guidelines for pediatric obesity were released in 2004 by Indian Academy of Pediatrics (IAP). Since then, there has been an alarming increase in prevalence and a significant shift in our understanding in the pathogenesis, risk factors, evaluation, and management of pediatric obesity and its complications. Thus, it was decided to revise and update the previous recommendations. OBJECTIVES: To review the existing literature on the burden of childhood obesity and its underlying etiology and risk factors. To recommend evaluation of childhood obesity and suggest optimum prevention and management strategies of childhood obesity. PROCESS: The following IAP chapters (Pediatric and Adolescent Endocrinology, Infant and Young Child feeding, Nutrition, Non-Communicable Disease and Adolescent Health Academy) were invited to nominate members to become part of the writing committee. The Committee held discussions on various aspects of childhood obesity through online meetings between February and August, 2023. Recommendations were then formulated, which were analyzed, revised and approved by all members of the Committee. RECOMMENDATIONS: Exogenous or primary obesity accounts for the majority of cases of childhood obesity. It is important to differentiate it from endogenous or secondary obesity as evaluation and management changes depending on the cause. In Indian, in children under 5 years of age, weight for length/height using WHO charts, and in children 5-18 years, BMI using IAP 2015 charts is used to diagnose overweight and obesity. Waist circumference should be routinely measured in all overweight and obese children and plotted on India specific charts, as it is a key measure of cardio-metabolic risk. Routine evaluation for endocrine causes is not recommended, except in short and obese children with additional diagnostic clues. All obese children more than ten years old should be evaluated for comorbidities like hypertension, dyslipidemia, hyperglycemia and non-alcoholic fatty liver disease/metabolic dysfunction associated steatotic liver disease (NAFLD/ MASLD). Prevention and management of childhood obesity mainly involves healthy diet practices, daily moderate to vigorous physical activity and reduced screen time. Pharmacotherapy may be offered as an addition to lifestyle interventions only in cases of class 3 obesity or if there are any life-threatening comorbidities. Finally, surgical management may be offered in children older than 12 years of age with class 2 obesity and associated comorbidities or class 3 obesity with/without comorbidities, only after failure of a proper trial of intense lifestyle modifications and pharmacotherapy for at least 6 months.

Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is facilitated by a web of signalling pathways constantly modulated by a multitude of proteins that regulate the flow of genetic information inside the pathogen. Transcription factors (TFs) belongs to one such family of proteins that modulate the signalling by regulating the abundance of proteins at the transcript level. In the current study, we have characterized the putative transcriptional regulatory protein encoded by the Rv1719 gene of Mycobacterium tuberculosis. This TF belongs to the IclR family of proteins with orthologs found in both bacterial and archaeal species. We cloned the Rv1719 gene into the pET28a expression vector and performed heterologous expression of the recombinant protein with E coli as the host. Further, optimization of the purification protocol by affinity chromatography and characterization of proteins for their functional viability has been demonstrated using various biochemical and/or biophysical approaches. Scale-up of purification yielded approximately 30 mg of ~ 28 kDa protein per litre of culture. In-silico protein domain analysis of Rv1719 protein predicted the presence of the helix-turn-helix (HTH) domain suggesting its ability to bind DNA sequence and modulate transcription; a hallmark of a transcriptional regulatory protein. Further, by performing electrophoretic mobility shift assay (EMSA) we demonstrated that the protein binds to a specific DNA fragment harboring the probable binding site of one of the predicted promoters.

Breastfeeding in Coronavirus Disease 2019 (COVID-19): Position Statement of Indian Academy of Pediatrics and Infant and Young Child Feeding Chapter.

JUSTIFICATION: Recent research has provided evidence for lack of transmission of SARS-CoV-2 through human milk and breastfeeding. Updating the practice guidelines will help in providing appropriate advice and support regarding breastfeeding during the coronavirus 2019 (COVID-19) pandemic. OBJECTIVES: To provide evidence-based guidelines to help the healthcare professionals to advise optimal breastfeeding practices during the COVID-19 pandemic. PROCESS: Formulation of key questions was done under the chairmanship of President of the IAP. It was followed by review of literature and the recommendations of other international and national professional bodies. Through Infant and Young child (IYCF) focused WhatsApp group opinion of all members was taken. The final document was prepared after the consensus and approval by all members of the committee. RECOMMENDATIONS: The IYCF Chapter of IAP strongly recommends unabated promotion, protection and support to breastfeeding during the COVID-19 pandemic with due precautions.

Mitochondrial Control of Stem Cell State and Fate: Lessons From Drosophila.

Over the years, Drosophila has served as a wonderful genetically tractable model system to unravel various facets of tissue-resident stem cells in their microenvironment. Studies in different stem and progenitor cell types of Drosophila have led to the discovery of cell-intrinsic and extrinsic factors crucial for stem cell state and fate. Though initially touted as the ATP generating machines for carrying various cellular processes, it is now increasingly becoming clear that mitochondrial processes alone can override the cellular program of stem cells. The last few years have witnessed a surge in our understanding of mitochondria's contribution to governing different stem cell properties in their subtissular niches in Drosophila. Through this review, we intend to sum up and highlight the outcome of these in vivo studies that implicate mitochondria as a central regulator of stem cell fate decisions; to find the commonalities and uniqueness associated with these regulatory mechanisms.

Fatty acid ß-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila.

Cell-intrinsic and extrinsic signals regulate the state and fate of stem and progenitor cells. Recent advances in metabolomics illustrate that various metabolic pathways are also important in regulating stem cell fate. However, our understanding of the metabolic control of the state and fate of progenitor cells is in its infancy. Using Drosophila hematopoietic organ: lymph gland, we demonstrate that Fatty Acid Oxidation (FAO) is essential for the differentiation of blood cell progenitors. In the absence of FAO, the progenitors are unable to differentiate and exhibit altered histone acetylation. Interestingly, acetate supplementation rescues both histone acetylation and the differentiation defects. We further show that the CPT1/whd (withered), the rate-limiting enzyme of FAO, is transcriptionally regulated by Jun-Kinase (JNK), which has been previously implicated in progenitor differentiation. Our study thus reveals how the cellular signaling machinery integrates with the metabolic cue to facilitate the differentiation program.

Stem cells are special precursor cells, found in all animals from flies to humans, that can give rise to all the mature cell types in the body. Their job is to generate supplies of new cells wherever these are needed. This is important because it allows damaged or worn-out tissues to be repaired and replaced by fresh, healthy cells. As part of this renewal process, stem cells generate pools of more specialized cells, called progenitor cells. These can be thought of as half-way to maturation and can only develop in a more restricted number of ways. For example, so-called myeloid progenitor cells from humans can only develop into a specific group of blood cell types, collectively termed the myeloid lineage. Fruit flies, like many other animals, also have several different types of blood cells. The fly's repertoire of blood cells is very similar to the human myeloid lineage, and these cells also develop from the fly equivalent of myeloid progenitor cells. These progenitors are found in a specialized organ in fruit fly larvae called the lymph gland, where the blood forms. These similarities between fruit flies and humans mean that flies are a good model to study how myeloid progenitor cells mature. A lot is already known about the molecules that signal to progenitor cells how and when to mature. However, the role of metabolism ­ the chemical reactions that process nutrients and provide energy inside cells ­ is still poorly understood. Tiwari et al. set out to identify which metabolic reactions myeloid progenitor cells require and how these reactions might shape the progenitors' development into mature blood cells. The experiments in this study used fruit fly larvae that had been genetically altered so that they could no longer perform key chemical reactions needed for the breakdown of fats. In these mutant larvae, the progenitors within the lymph gland could not give rise to mature blood cells. This showed that myeloid progenitor cells need to be able to break down fats in order to develop properly. These results highlight a previously unappreciated role for metabolism in controlling the development of progenitor cells. If this effect also occurs in humans, this knowledge could one day help medical researchers engineer replacement tissues in the lab, or even increase our own bodies' ability to regenerate blood, and potentially other organs.

Prediction of Small Molecule Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus-2 RNA-dependent RNA Polymerase.

The current COVID-19 outbreak warrants the design and development of novel anti-COVID therapeutics. Using a combination of bioinformatics and computational tools, we modelled the 3D structure of the RdRp (RNA-dependent RNA polymerase) of SARS-CoV2 (severe acute respiratory syndrome coronavirus-2) and predicted its probable GTP binding pocket in the active site. GTP is crucial for the formation of the initiation complex during RNA replication. This site was computationally targeted using a number of small molecule inhibitors of the hepatitis C RNA polymerase reported previously. Further optimizations suggested a lead molecule that may prove fruitful in the development of potent inhibitors against the RdRp of SARS-CoV2.

Prevention of Micronutrient Deficiencies in Young Children: Consensus Statement from Infant and Young Child Feeding Chapter of Indian Academy of Pediatrics.

JUSTIFICATION: Micronutrient deficiencies have significant impact on the overall health and well-being of society and potential targets for supplementations. It is important to formulate a consensus statement in view of current evidence, and put in place strategies to meet targets. OBJECTIVE: To formulate by endorsement or adoption and disseminate a consensus statement for prevention of micronutrients deficiencies in young children for office practices from an Indian perspective. PROCESS: A National Consultative Meeting was convened by Infant and Young Child Feeding Chapter (IYCF) of Indian Academy of Pediatrics (IAP) on 17 December, 2016 at Mumbai. IYCF chapter, IAP, United Nations Children Fund, National Institute of Nutrition and Government of India were the participating agencies; and participants representing different parts of India were included. CONCLUSIONS: Micronutrient deficiencies are widespread. For its prevention proper maternal and infant-young child feeding strategies need to be practiced. Encourage delayed cord clamping, dietary diversification, germinated foods, soaking and fermentation processes. Existing Iron, Vitamin A, Zinc supplementation and universal salt iodization programs need to be scaled up, especially in high risk groups. Universal vitamin D supplementation need to be in place; though, the dose needs more research. Vitamin B12 deficiency screening and supplementation should be practiced only in high-risk groups. Availability of appropriately fortified foods needs to be addressed urgently.

End-of-Life Care: Consensus Statement by Indian Academy of Pediatrics.

JUSTIFICATION: The right to life has been accepted as one of the fundamental rights in our constitution. Resuscitation is a procedure performed for all patients suffering from cardiac or respiratory arrest irrespective of the clinical condition. There are no legal guidelines defining process to be adopted in situations where resuscitation is unlikely to be useful. There are no guidelines on withdrawal of care or end of life (EOL) decisions, accepted by the Government, judiciary, professionals, academicians or the community. PROCESS: A National Consultative meet was organized by Indian Medico-Legal and Ethics Association and the Medico-legal group of Indian Academy of Pediatrics (IAP) to formulate the guidelines on 'Do Not Resuscitate' (DNR), and 'End of Life Support'. The meeting was organized on 30th May, 2014 at Ram Manohar Lohia Hospital, New Delhi. The meeting involved professionals from legal and various medical fields as well as administrators, and members from Medical Council of India. OBJECTIVES: To frame the guidelines related to EOL care issues and withdrawal or with-holding treatment in situations where outcome of continued treatment is expected to be poor in terms of ultimate survival or quality of life. RECOMMENDATIONS: (i) DNR or end of life care should not be activated till consensus is achieved between treating team and the next of kin; (ii) Consensus within health care team (including nurses) needs to be achieved before discussion with family members; (iii) Discussion should involve the family members - next of kin and other persons who can influence decisions; (iv) If family members want to include their family physician or a prominent person from the community, it should be encouraged. Similarly if family members want a particular member of treating team, he/she should be included; (v) Treating doctors should have all the facts of the case including investigations available with them before discussion; (vi) Unit in-charge or treating doctor should be responsible for achieving consensus and should initiate the discussion; (vii) After presenting the facts of the cases, family members should be encouraged to ask questions and clear doubts (if any); (viii) At the end of discussion, a summary of the discussion should be prepared and signed by the next of kin and the unit in-charge or treating doctors; (ix) DNR orders should be reviewed in the event of unexpected improvement or on request of next of kin. Same should be documented; (x) DNR orders remain valid during transport.

Infant and Young Child Feeding Guidelines, 2016.

JUSTIFICATION: Shaping up the post-2015 development agenda is of crucial importance in the development process around the Globe as 2015 was the last year of milllionium development goals. It is the right time to asses our own progress vis-a-vis the Millennium Development Goals and these Guidelines are an attempt in that regard. PROCESS: The Infant and Young Child Feeding (IYCF) chapter of Indian Academy of Pediatrics invited a group of experts for National Consultative Meet for discussing and contributing on latest scientific advances and developments. Various partners from WHO, UNICEF, Ministry of Child Welfare Department, Ministry of Health and Family Welfare, Ministry of Chemical and Fertilizers of Govt of India, Human Milk Banking Association (of India), Indian Medico-Legal and Ethics Association (IMLEA), non-governmental organizations and academicians from various states of India contributed to these guidelines. The guidelines were finalized during the IYCNCON 2015 at New Delhi in August 2015. OBJECTIVES: To formulate, endorse, adopt and disseminate guidelines related to Infant and Young Child feeding from an Indian perspective (including human milk banking, infant feeding in the HIV situation, and micro-nutrients). RECOMMENDATIONS: Early initiation of breastfeeding within first hour of birth, exclusive breastfeeding for the first six months followed by continued breastfeeding for up to two years and beyond with appropriate complementary foods after completion of 6 months is the most appropriate feeding strategy. Micro-nutrient supplementation in infants, and adequate nutrition and anemia control for adolescent girls, pregnant and lactating mothers is advocated. Concepts and need for human milk banks in India has also been incorporated.

The IMS Act 1992: need for more amendments and publicity.

The IMS act was passed after a lot of thinking, discussions over pros and cons of childhood feeding practices and recommendations of WHA. We are not only legally bound by it, but it is also our moral responsibility to see that the act works. Already 9-10 years have passed but the results are not that encouraging. Most of our colleagues are unaware of the provisions and importance of this act. Making the law effective is very time consuming, costly, unaffordable and avoidable affair. We should not harp on multiplicity of legal opinions & loopholes to allow breaking the law in spirit if not in letter. We feel that most of our laws are paper tigers without any teeth, gathering dust by remaining present only in law books or journals. If we don't act now then there is every possibility that this act may also become one of the historical legal documents. Hence it is time for all of us to become an activist or counselor for BF. We should create awareness and public opinion about protection, promotion and support for BF. Be vigilant, form community watch group and report violations to the authorities. So that our country can become a proud nation of healthy, intelligent and emotionally stable children in the 21st century.

Human milk banking guidelines.

JUSTIFICATION: WHO and UNICEF state that the use of human milk from other sources should be the first alternative when it is not possible for the mother to breastfeed. Human milk banks should be made available in appropriate situations. The IYCF Chapter is actively concerned about the compelling use of formula feeds in the infants because of the non availability of human breast milk banks. PROCESS: A National Consultative Meet for framing guidelines was summoned by the IYCF Chapter and the Ministry of Health and Family Welfare, Government of India on 30th June, 2013, with representations from various stakeholders. The guidelines were drafted after an extensive literature review and discussions. Though these guidelines are based on the experiences and guidelines from other countries, changes have been made to suit the Indian setup, culture and needs, without compromising scientific evidence. OBJECTIVES: To ensure quality of donated breast milk as a safe end product. RECOMMENDATIONS: Human Milk Banking Association should be constituted, and human milk banks should be established across the country. National coordination mechanism should be developed with a secretariat and technical support to follow-up on action in States. Budgetary provisions should be made available for the activities.

Evaluation of Cameroonian plants towards experimental bone regeneration.

ETHNOPHARMACOLOGICAL RELEVANCE: Elephantopus mollis, Spilanthes africana, Urena lobata, Momordica multiflora, Asystasia gangetica and Brillantaisia ovariensis are used in Cameroonian traditional medicine for the treatment of bone diseases and fracture repair. The aim of this study was to evaluate the effect of ethanolic extracts of six Cameroonian medicinal plants on bone regeneration following bone and marrow injury. MATERIALS AND METHODS: Ethanol extract of Cameroonian medicinal plants were administered (each extract at 250, 500 and 750mg/kg doses) orally to adult female Sprague-Dawley rats having a drill hole injury (0.8mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of fractured bone was performed to evaluate bone regeneration (calcein labeling). Only active plant extracts were used for further experiments. Thus, callus was analyzed by microcomputed tomography. Osteogenic effects of the extracts were evaluated by assessing mineralized nodules formation of bone marrow stromal cells and osteoblast recruitment at drill hole site by immunohistochemistry. RESULTS: Ethanolic extract of the leaves and twigs of Elephantopus mollis (EM) and whole plant of Spilanthes africana (SA) dose-dependently stimulated bone regeneration at the drill hole site. EM at 250 and 750mg/kg doses and SA at 750mg/kg dose significantly increased mineral deposition compared to controls. Both extracts at 500 and 750mg/kg doses improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. EM and SA extracts increased the formation of mineralized nodules from the bone marrow stromal cells. In addition, EM and SA extracts increased osteoblast recruitment at the drill hole site evident from increased Runx-2 positive cells following their treatments compared to control. CONCLUSION: Ethanolic extracts of EM and SA accelerate fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying their traditional use.