Automated, image-based quantification of peroxisome characteristics with perox-per-cell.

perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually-quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. The software is available at https://github.com/AitchisonLab/perox-per-cell.

Aeroplane wing, a new recessive autosomal phenotypic marker in the malaria vector, Anopheles stephensi Liston.

A novel and distinct mutant with a phenotype, aeroplane wing (ae) is reported for the first time in the urban malaria vector Anopheles stephensi. The main aim of this study was to establish the mode of inheritance of the ae gene performing genetic crossings between the mutants and wild types. These mutants show extended open wings that are visible to naked eyes in both the sexes. Mutants were first noticed in a nutritionally stressed isofemale colony. Strategic genetic crosses revealed that the ae gene is a recessive, autosomal, and monogenic trait having full penetrance with uniform expression in its adult stage. Egg morphometric analysis confirmed that these mutants were intermediate variant. No significant differences were observed in the wing venation and size of ae mutants compared to their control parental lines. Further cytogenetic analysis on the ovarian polytene chromosome of ae mutant showed an inversion (3Li) on the 3L arm like its parental line. This ae mutant would be a prominent marker and could be useful to study the functions of related specific genes within its genome.

Convergent and divergent mechanisms of peroxisomal and mitochondrial division.

Organelle division and segregation are important in cellular homeostasis. Peroxisomes (POs) and mitochondria share a core division machinery and mechanism of membrane scission. The division of each organelle is interdependent not only on the other but also on other organelles, reflecting the dynamic communication between subcellular compartments, even as they coordinate the exchange of metabolites and signals. We highlight common and unique mechanisms involved in the fission of these organelles under the premise that much can be gleaned regarding the division of one organelle based on information available for the other.

Enrichment of phenotype among biological forms of Anopheles stephensi Liston through establishment of isofemale lines.

BACKGROUND: Vector management programs rely on knowledge of the biology and genetic make-up of mosquitoes. Anopheles stephensi is a major invasive urban malaria vector, distributed throughout the Indian subcontinent and Middle East, and has recently been expanding its range in Africa. With the existence of three biological forms, distinctly identifiable based on the number of ridges on eggs and varying vectorial competence, An. stephensi is a perfect species for developing isofemale lines, which can be tested for insecticide susceptibility and vectorial competence of various biological forms. METHODS: We describe key steps involved in establishment and validation of isofemale lines. Isofemale colonies were further used for the characterization of insecticide susceptibility and differential vector competence. The results were statistically evaluated through descriptive and inferential statistics using Vassar Stat and Prism GraphPad software packages. RESULTS: Through a meticulous selection process, we overcame an initial inbreeding depression and found no significant morphometric differences in wings and egg size between the parental and respective isofemale lines in later generations. IndCh and IndInt strains showed variations in resistance to different insecticides belonging to all four major classes. We observed a significant change in vectorial competence between the respective isofemale and parental lines. CONCLUSIONS: Isofemale lines can be a valuable resource for characterizing and enhancing several genotypic and phenotypic traits. This is the first detailed report of the establishment of two isofemale lines of type and intermediate biological forms in Anopheles stephensi. The work encompasses characterization of fitness traits among two lines through a transgenerational study. Furthermore, isofemale colonies were established and used to characterize insecticide susceptibility and vector competence. The study provides valuable insights into differential susceptibility status of the parental and isofemale lines to different insecticides belonging to the same class. Corroborating an earlier hypothesis, we demonstrate the high vector competence of the type form relative to the intermediate form using homozygous lines. Using these lines, it is now possible to study host-parasite interactions and identify factors that might be responsible for altered susceptibility and increased vector competence in An. stephensi biological forms that would also pave the way for developing better vector management strategies.

Identification of a TNF-TNFR-like system in malaria vectors (Anopheles stephensi) likely to influence Plasmodium resistance.

Identification of Plasmodium-resistance genes in malaria vectors remains an elusive goal despite the recent availability of high-quality genomes of several mosquito vectors. Anopheles stephensi, with its three distinctly-identifiable forms at the egg stage, correlating with varying vector competence, offers an ideal species to discover functional mosquito genes implicated in Plasmodium resistance. Recently, the genomes of several strains of An. stephensi of the type-form, known to display high vectorial capacity, were reported. Here, we report a chromosomal-level assembly of an intermediate-form of An. stephensi strain (IndInt), shown to have reduced vectorial capacity relative to a strain of type-form (IndCh). The contig level assembly with a L50 of 4 was scaffolded into chromosomes by using the genome of IndCh as the reference. The final assembly shows a heterozygous paracentric inversion, 3Li, involving 8 Mbp, which is syntenic to the extensively-studied 2La inversion implicated in Plasmodium resistance in An. gambiae involving 21 Mbp. Deep annotation of genes within the 3Li region in the IndInt assembly using the state-of-the-art protein-fold prediction and other annotation tools reveals the presence of a tumor necrosis factor-alpha (TNF-alpha) like gene, which is the homolog of the Eiger gene in Drosophila. Subsequent chromosome-wide searches revealed homologs of Wengen (Wgn) and Grindelwald (Grnd) genes, which are known to be the receptors for Eiger in Drosophila. We have identified all the genes in IndInt required for Eiger-mediated signaling by analogy to the TNF-alpha system, suggesting the presence of a functionally-active Eiger signaling pathway in IndInt. Comparative genomics of the three type-forms with that of IndInt, reveals structurally disruptive mutations in Eiger gene in all three strains of the type-form, suggesting compromised innate immunity in the type-form as the likely cause of high vectorial capacity in these strains. This is the first report of the presence of a homolog of Eiger in malaria vectors, known to be involved in cell death in Drosophila, within an inversion region in IndInt syntenic to an inversion associated with Plasmodium resistance in An. gambiae.

OXPHOS deficiencies affect peroxisome proliferation by downregulating genes controlled by the SNF1 signaling pathway.

How environmental cues influence peroxisome proliferation, particularly through organelles, remains largely unknown. Yeast peroxisomes metabolize fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, produced by these pathways enter mitochondria for ATP production and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH-shuttling- and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by a high AMP/ATP ratio. In OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Δgal83 mutant, were rescued by deletion of three transcriptional repressor genes (MIG1, MIG2, and NRG1) controlled by SNF1 signaling. Our results are interpreted in terms of a mechanism by which peroxisomal and mitochondrial proteins and/or metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol, and the nucleus. We discuss the physiological relevance of this work in the context of human OXPHOS deficiencies.

The genome trilogy of Anopheles stephensi, an urban malaria vector, reveals structure of a locus associated with adaptation to environmental heterogeneity.

Anopheles stephensi is the most menacing malaria vector to watch for in newly urbanising parts of the world. Its fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions in the genome of malaria vectors are implicated in adaptation. We report the genome assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome reported here with a L50 of 4, completes the trilogy of high-resolution genomes of strains with respect to a 16.5 Mbp 2Rb genotype in An. stephensi known to be associated with adaptation to environmental heterogeneity. Unlike the reported genomes of two other strains, STE2 (2R+b/2Rb) and UCI (2Rb/2Rb), IndCh is found to be homozygous for the standard form (2R+b/2R+b). Comparative genome analysis revealed base-level details of the breakpoints and allowed extraction of 22,650 segregating SNPs for typing this inversion in populations. Whole genome sequencing of 82 individual mosquitoes from diverse geographical locations reveal that one third of both wild and laboratory populations maintain the heterozygous genotype of 2Rb. The large number of SNPs can be tailored to 1740 exonic SNPs enabling genotyping directly from transcriptome sequencing. The genome trilogy approach accelerated the study of fine structure and typing of an important inversion in An. stephensi, putting the genome resources for this understudied species on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse geographical locations cluster with IndCh, pointing to significant convergence resulting from travel and commerce between cities, perhaps, contributing to the survival of the fittest strain.

Recognition and Chaperoning by Pex19, Followed by Trafficking and Membrane Insertion of the Peroxisome Proliferation Protein, Pex11.

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation.

BiFC Method Based on Intraorganellar Protein Crowding Detects Oleate-Dependent Peroxisomal Targeting of Pichia pastoris Malate Dehydrogenase.

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly.

BACKGROUND: The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector's genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution. RESULTS: To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role. CONCLUSION: Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.

Balancing the Opposing Principles That Govern Peroxisome Homeostasis.

Despite major advances in our understanding of players and mechanisms involved in peroxisome biogenesis and peroxisome degradation, very few studies have focused on unraveling the multi-layered connections between, and the coordination of, these two opposing processes that regulate peroxisome homeostasis. The intersection between these processes also provides exciting avenues for future research. This review highlights the links between peroxisome biogenesis and degradation, incorporating an integrative approach that is critical not only for a mechanistic understanding, but also for manipulating the balance between these processes in relevant disease models.

A Near-Chromosome Level Genome Assembly of Anopheles stephensi.

Malaria remains a major healthcare risk to growing economies like India, and a chromosome-level reference genome of Anopheles stephensi is critical for successful vector management and understanding of vector evolution using comparative genomics. We report chromosome-level assemblies of an Indian strain, STE2, and a Pakistani strain SDA-500 by combining draft genomes of the two strains using a homology-based iterative approach. The resulting assembly IndV3/PakV3 with L50 of 9/12 and N50 6.3/6.9 Mb had scaffolds long enough for building 90% of the euchromatic regions of the three chromosomes, IndV3s/PakV3s, using low-resolution physical markers and enabled the generation of the next version of genome assemblies, IndV4/PakV4, using HiC data. We have validated these assemblies using contact maps against publicly available HiC raw data from two strains including STE2 and another lab strain of An. stephensi from UCI and compare the quality of the assemblies with other assemblies made available as preprints since the submission of the manuscript. We show that the IndV3s and IndV4 assemblies are sensitive in identifying a homozygous 2Rb inversion in the UCI strain and a 2Rb polymorphism in the STE2 strain. Multiple tandem copies of CYP6a14, 4c1, and 4c21 genes, implicated in insecticide resistance, lie within this inversion locus. Comparison of assembled genomes suggests a variation of 1 in 81 positions between the UCI and STE2 lab strains, 1 in 82 between SDA-500 and UCI strain, and 1 in 113 between SDA-500 and STE2 strains of An. stephensi, which are closer than 1 in 68 variations among individuals from two other lab strains sequenced and reported here. Based on the developmental transcriptome and orthology of all the 54 olfactory receptors (ORs) to those of other Anopheles species, we identify an OR with the potential for host recognition in the genus Anopheles. A comparative analysis of An. stephensi genomes with the completed genomes of a few other Anopheles species suggests limited inter-chromosomal gene flow and loss of synteny within chromosomal arms even among the closely related species.

Axis vertebral dimensions for safe screw placement: A CT normative data analysis.

INTRODUCTION: Morphometric evaluation of the pedicle and isthmus of second cervical vertebra (C2) (Axis) is extremely vital before contemplating any surgical stabilization involving the Craniovertebral region, in view of its proximity to the vertebral artery and the cervical nerve root. The dimensions of pedicles and isthmuses in C2 vary between individuals and there is paucity of data in the Indian population. This study strives to measure the average pedicle and isthmus dimensions in a sample of population, which would enable selection of screws with safest diameters to be used in C2; thereby avoiding injury to adjacent neurovascular structures. MATERIALS AND METHODS: One Hundred patients in the age group between 18 and 70 years who underwent CT scan of head and neck region were included in the study. The aim of this study was to assess the anatomic suitability of transarticular and pedicle screw placement in Axis vertebrae of Indian population and determine the maximum safe diameter for screw placement. The following parameters were measured in millimeters: Pedicle width, Pedicle angle, Internal height and Isthmic height. RESULTS: The Mean maximum diameter of potential pedicle screw was 4.99 ± 1.1 mm for the right side with the left side being slightly wider at 5.20 ± 1.16 mm. Twenty eight (28%; 56 out of 200 pedicles) had a measurement < 4.5 mm. The internal height in sagittal images representing the pedicle height was found to be 4.79 ± 0.96 mm on the right side and 4.75 ± 1.04 mm on the left side. Sixty five (65) out of 200 pedicles (32.5%) had measurements < 4.5 mm in sagittal plane. The Mean maximum diameter of potential Transarticular screw (outer diameter of isthmus) was 5.05 ± 0.78 mm for the right side and 5.18 ± 0.84 mm on the left side. DISCUSSION: Isthmic height < 4.5 mm could potentially violate the vertebral foramen when a 3.5 mm screw is used. In our study 22.5% isthmuses were narrow (<4.5 mm). The mean maximum safe diameter for a potential transarticular screw in the present study was 5.11 mm. Though our patients had smaller isthmus dimensions compared with literature, 77.5% of C2 could take a 4 mm transarticular screw quite comfortably considering the 0.5 mm margin on either side. In the present study, 28% of pedicles were found to be inappropriately sized (<4.5 mm) to accommodate the standard 3.5 mm screw. The mean maximum diameter of a potential pedicle screw in our study was 5.09 mm; and in 72% of patients a 4 mm screw could be placed with confidence. Though our patients on an average can accommodate a 4 mm screw comfortably, we suggest a protocol of obtaining CT measurements of C2 prior to operative intervention for identifying those individuals at risk of neurovascular injury; 22.5% for transarticular screw and 28% for pedicle screw.

Rare Case Report of Closed Traumatic Dislocation of Second to Fifth Metatarsophalangeal Joints.

Closed traumatic dislocation of multiple metatarsophalangeal joints is a rare injury. Until now only one case of simultaneous dislocation of all five metatarsophalangeal joints has been reported in peer-reviewed studies. The complex anatomy of the metatarsophalangeal joints prevents the relocation of the joints in a closed manner in maximum cases. We are reporting a case of dorsal dislocation of the second to fifth metatarsophalangeal joints in the left foot after road traffic accident. Bony prominence over the plantar aspect and increased web space between toes on presentation, then incongruity of metatarsophalangeal joints has to be thoroughly checked on radiograph. Since closed reduction attempts failed open reduction was done through dorsal approach using two incisions. Button holing of the capsule with interposition of capsule and plantar plate was noted. Dorsal approach avoids damage to the plantar plate and surrounding soft tissues.

Mechanistic Insights into the Role of Atg11 in Selective Autophagy.

Macroautophagy (referred to hereafter as autophagy) is an intracellular degradation pathway in which the formation of a double-membrane vesicle called the autophagosome is a key event in the transport of multiple cytoplasmic cargo (e.g., proteins, protein aggregates, lipid droplets or organelles) to the vacuole (lysosome in mammals) for degradation and recycling. During this process, autophagosomes are formed de novo by membrane fusion events leading to phagophore formation initiated at the phagophore assembly site. In yeast, Atg11 and Atg17 function as protein scaffolds, essential for selective and non-selective types of autophagy, respectively. While Atg17 functions in non-selective autophagy are well-defined in the literature, less attention is concentrated on recent findings regarding the roles of Atg11 in selective autophagy. Here, we summarize current knowledge about the Atg11 scaffold protein and review recent findings in the context of its role in selective autophagy initiation and autophagosome formation.

The autophagic degradation of cytosolic pools of peroxisomal proteins by a new selective pathway.

Damaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for Pichia pastoris and Atg36 for Saccharomyces cerevisiae, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In P. pastoris, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products.Abbreviations: ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation.

Late-onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5S163R/wt ), and homozygous knock-in (Ctrp5S163R/S163R ) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology.

Factors affecting compliance to hospital visit among clubfoot patients: A cross-sectional study from a tertiary referral clubfoot clinic in the developing country.

PURPOSE: Ensuring compliance to treatment protocol, especially regular visit to treating facility, is an important aspect of clubfoot management. However, the factors affecting compliance to follow-up schedule are myriad. METHODS: A cross-sectional study was undertaken among caregivers of clubfoot patients from a tertiary referral clubfoot clinic in a developing country. Hospital records were reviewed to collect demographic data and subjects were classified as either "regular" or "irregular" if they missed ≤3 and >3 scheduled hospital visits, respectively. Various factors that could affect compliance such as family size, number of children, literacy of caregiver, occupation of breadwinner, and time taken to travel to hospital were studied. Caregivers were probed regarding the reason for their irregularity. RESULTS: A total of 238 patients were included, of which 138 formed the "regular" group and the rest 100 formed the "irregular" group. Patients in the regular group were significantly younger (mean age 43.8 months) compared to the irregular group (59.8 months; p = 0.001). The mean follow-up period in the regular group was 28.1 months and in the irregular group was 33.8 months. On univariate analysis, age, duration of follow-up, and transport duration were found to be significant between the two groups. However, multivariate analysis revealed that female children with clubfoot are more likely to be irregular as compared to males ( p = 0.038). CONCLUSION: In a developing country setting, higher age and being a female child are associated with irregularity to hospital visit protocol. At clubfoot clinics, identifying these children and counseling their caregivers might improve compliance.