Protocol for evaluating mitochondrial morphology changes in response to CCCP-induced stress through open-source image processing software.

Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al.1.

PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics.

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.

Three-year review of a capacity building pilot for a sustainable regional network on food, nutrition and health systems education in India.

BACKGROUND: In Kolkata (India), there are high rates of malnourished children (45.9%) under the age of three, impacting growth, organ development, function, and cognition. Mothers have a major role to play during this crucial development stage, with research showing nutrition knowledge, attitudes and practices (KAP) of mothers are important determinants of childhood malnutrition. AIMS: To document 3 years of capacity building towards a sustainable nutrition education network in Kolkata, India, while assessing the ability to perform data collection in the form of needs assessments, impact assessments and capacity reviews. METHODS: Descriptive review and analysis of engagement and impact from 3 years of work by the NNEdPro Global Centre for Nutrition and Health, initiating locally led nutrition education interventions. Mapping to the Indian National Nutrition Strategy was also performed to review adherence to nationwide priorities surrounding nutrition and determine the wider application potential of the network. RESULTS: Two simultaneous projects were taken forward by a team of local healthcare professionals and student champions. Project 1-medical college workshops for medical student nutrition education with added focus on underserved populations, Project 2-preparation for a 'Mobile Teaching Kitchen' (MTK) in marginalised communities to empower local women as nutrition educators.Data collection methods used for analysing markers of impact and sustainability were semi-structured interviews of the community members, and KAP questionnaires to assess response to educational sessions. CONCLUSION: With local support it is possible to create and sustain fieldwork for an extended period with meaningful outputs and impact. This initiative demonstrates that it is possible to use healthcare professionals, students and volunteers with low-intensity training and a low-cost approach to produce action research with considerable impact and results in rapid, reliable and robust manner.

'Bhavishya Shakti: Empowering the Future': establishing and evaluating a pilot community mobile teaching kitchen as an innovative model, training marginalised women to become nutrition champions and culinary health educators in Kolkata, India.

BACKGROUND: Malnutrition is a global emergency, creating an overlapping burden on individual, public and economic health. The double burden of malnutrition affects approximately 2.3 billion adults worldwide. Following 3 years of capacity building work in Kolkata, with assistance of local volunteers and organisations, we established an empowering nutrition education model in the form of a 'mobile teaching kitchen (MTK)' with the aim of creating culinary health educators from lay slum-dwelling women. AIMS: To evaluate the piloting of a novel MTK nutrition education platform and its effects on the participants, alongside data collection feasibility. METHODS: Over 6 months, marginalised (RG Kar and Chetla slums) women underwent nutrition training using the MTK supported by dietitians, doctors and volunteers. Preintervention and postintervention assessments of knowledge, attitudes and practices (KAP), as well as anthropometric and clinical nutritional status of both the women and their children were recorded. The education was delivered by a 'See One, Do One, Teach One' approach with a final assessment of teaching delivery performed in the final session. RESULTS: Twelve women were trained in total, six from each slum. Statistically significant improvements were noted in sections of KAP, with improvements in nutrition knowledge (+4.8) and practices (+0.8). In addition, statistically significant positive changes were seen in 'understanding of healthy nutrition for their children' (p=0.02), 'sources of protein rich food' (p=0.02) and 'not skipping meals if a child is ill' (p≤0.001). CONCLUSION: The MTK as a public health intervention managed to educate, empower and upskill two groups of lay marginalised women into MTK Champions from the urban slums of Kolkata, India. Improvements in their nutrition KAP demonstrate just some of the effects of this programme. By the provision of healthy meals and nutritional messages, the MTK Champions are key drivers nudging improvements in nutrition and health related awareness with a ripple effect across the communities that they serve. There is potential to upscale and adapt this programme to other settings, or developing into a microenterprise model, that can help future MTK Champions earn a stable income.

Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles.

Sorting endosomes (SEs) are the regulatory hubs for sorting cargo to multiple organelles, including lysosome-related organelles, such as melanosomes in melanocytes. In parallel, melanosome biogenesis is initiated from SEs with the processing and sequential transport of melanocyte-specific proteins toward maturing melanosomes. However, the mechanism of cargo segregation on SEs is largely unknown. Here, RNAi screening in melanocytes revealed that knockdown of Rab4A results in defective melanosome maturation. Rab4A-depletion increases the number of vacuolar endosomes and disturbs the cargo sorting, which in turn lead to the mislocalization of melanosomal proteins to lysosomes, cell surface and exosomes. Rab4A localizes to the SEs and forms an endosomal complex with the adaptor AP-3, the effector rabenosyn-5 and the motor KIF3, which possibly coordinates cargo segregation on SEs. Consistent with this, inactivation of rabenosyn-5, KIF3A or KIF3B phenocopied the defects observed in Rab4A-knockdown melanocytes. Further, rabenosyn-5 was found to associate with rabaptin-5 or Rabip4/4' (isoforms encoded by Rufy1) and differentially regulate cargo sorting from SEs. Thus, Rab4A acts a key regulator of cargo segregation on SEs.This article has an associated First Person interview with the first author of the paper.

Visualization of Intracellular Tyrosinase Activity in vitro.

Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we described a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.