Recommendations for standard criteria for the positional and morphological evaluation of temporomandibular joint osseous structures using cone-beam CT: a systematic review.

OBJECTIVE: This systematic review aimed to appraise the reliability and comprehensiveness of imaging methods in studies that used three-dimensional assessment of the temporomandibular joint (TMJ) in order to propose a standardized imaging method. METHODS: Six databases/search engines were searched up until September 2022. The outcomes of interest included measurements of the mandibular condyle, glenoid fossa, joint spaces, or the entire TMJ. Two checklists were utilized: one to assess the risk of bias, with a maximum score of 37, and the other, a pre-designed checklist consisting of 22 items to evaluate the comprehensiveness of the methods used, with a maximum score of 33. RESULTS: Out of the 2567 records retrieved, only 14 studies, which used cone bean computed tomography (CBCT), were deemed eligible and thus included in the qualitative analysis. Three studies were deemed of low risk of bias, while the remaining studies were rated as moderate to high risk of bias, primarily due to improper reporting of inter-observer agreement, varying reliability values, and a limited number of cases included in the reliability analysis. Regarding the comprehensiveness of the methods used, only four studies achieved relatively high scores. The deficiencies observed were related to the reporting of variables such as slice thickness and voxel size, absence of or improper reporting of intra- and inter-examiner reliability analyses, and failure to assess all osseous components of the TMJ. CONCLUSION: CBCT-based methods used to assess the positions and morphology of TMJ bony structures appear to be imperfect and lacking in comprehensiveness. Hence, criteria for a standardized assessment method of these TMJ structures are proposed. CLINICAL RELEVANCE STATEMENT: Accurately, comprehensively, and reliably assessing the osseous structures of the temporomandibular joint will provide valid and valuable diagnostic features of the normal temporomandibular joint, and help establish potential associations between these osseous features and temporomandibular disorders. REGISTRATION: The protocol for this systematic review was registered at the International Prospective Register of Systematic Reviews (PROSPERO, No.: CRD42020199792). KEY POINTS: •Although many methods have been introduced to assess the osseous structure of the temporomandibular joint, they yielded inconsistent findings. •None of the published studies comprehensively assessed the temporomandibular joint. •Recommendations for a comprehensive temporomandibular joint osseous assessment method were suggested for better validity and reliability of future research.

Periodontal-Systemic Disease: A Study on Medical Practitioners' Knowledge and Practice.

OBJECTIVE: Oral health is intricately linked with systemic health. However, the knowledge and practice levels of medical practitioners (MPs) about this concern are extremely variable. The current study, therefore, sought to assess the status of knowledge and practice of MPs concerning the link between periodontal disease and different systemic conditions as well as the efficacy of a webinar as an interventional tool in enhancing knowledge of MPs of Jazan Province of Saudi Arabia. METHODS: This prospective interventional study involved 201 MPs. A 20-item questionnaire on evidence-based periodontal/systemic health associations was used. The participants answered the questionnaire before and 1 month after a webinar training that explained the mechanistic interrelation of periodontal and systemic health. McNemar test was performed for statistical analysis. RESULTS: Out of the 201 MPs who responded to the pre-webinar survey, 176 attended the webinar and hence were included in the final analyses. Sixty-eight (38.64%) were female, and 104 (58.09%) were older than 35 years. About 90% of MPs reported not being trained on oral health. Pre-webinar, 96 (54.55%), 63 (35.80%), and 17 (9.66%) MPs rated their knowledge about the association of periodontal disease with systemic diseases as limited, moderate, and good, respectively. Post-webinar, these figures improved remarkably: 36 (20.45%), 88 (50.00%), and 52 (29.55%) MPs rated their knowledge as limited, moderate, and good, respectively. Around 64% of MPs had relatively good levels of knowledge about the positive influence of periodontal disease treatment on diabetic patients' blood glucose levels. CONCLUSIONS: MPs revealed low levels of knowledge on the oral and systemic disease interrelationship. Conducting webinars on the oral-systemic health interrelationship seems to improve the overall knowledge and understanding of MPs.

Kinesio Taping as a Therapeutic Tool for Masticatory Myofascial Pain Syndrome-An Insight View.

Myofascial pain syndrome (MPS) is thought to stem from masticatory muscle hypersensitivity. Masticatory myofascial pain syndrome (MMPS) is characterized by multiple trigger points (MTrPs), also known as hyperirritable points, in taut bands of affected muscles, regional muscle pain, or referred pain to nearby maxillofacial areas like teeth, masticatory muscles or the temporomandibular joint (TMJ). Muscle stiffness, reduced range of motion, muscle weakening without atrophy, and autonomic symptoms may accompany regional discomfort. Multiple treatments have been utilized to reduce trigger points and mandibular function restrictions. As a result of these incapacitating symptoms, MMPS can significantly impair many elements of quality of life. The application of Kinesio tape (KT) is a non-invasive method of treating dormant myofascial trigger points. Utilizing the body's innate capacity for self-repair, this technique entails taping specific regions of the skin. KT alleviates discomfort, decreases swelling and inflammation, enhances or suppresses motor function in the muscles, stimulates proprioception, promotes lymphatic drainage, stimulates blood flow, and expedites tissue recovery. However, studies conducted to assess its effects have frequently yielded contradictory results. To the best of our knowledge, just a few research has looked into the therapeutic effects of KT on MMPS. The purpose of this review is to determine the efficacy of KT as a therapeutic tool for regular treatment or as an adjunct to existing therapy for MMPS based on the evidence presented in this review. To establish KT as a reliable independent treatment option, additional research is necessary to confirm the efficacy of KT techniques and applications, specifically randomized clinical trials.

Efficacy of lycopene for management of oral potentially malignant disorders: A systematic review and meta-analysis.

OBJECTIVE: The aim of this study was to investigate the available evidence on the efficacy of lycopene in the management of oral potentially malignant disorders (OPMDs). STUDY DESIGN: PubMed, Scopus, Web of Science, Google Scholar, China National Knowledge Infrastructure, and ProQuest databases were searched up to April 20, 2022. All clinical trials that assessed the efficacy of lycopene (I) on the signs/symptoms (O) of patients with OPMDs (P) in comparison to either active control or placebo (C) were included. Meta-analysis was conducted using the RevMan software (Cochrane Collaboration, London, UK). RESULTS: A total of 27 clinical trials (20 on oral submucosa fibrosis [OSF], 5 on oral lichen planus [OLP], and 2 on leukoplakia) were included. Overall, lycopene was efficacious in reducing signs and symptoms of OSF, OLP, and leukoplakia. The pooled data revealed comparable efficacy of lycopene and prednisolone in reducing pain and promoting clinical resolution of OLP. Additionally, the pooled data reported comparable efficacy of lycopene and conventional controls in improving the mouth opening and tongue protrusion in patients with OSF. CONCLUSIONS: The results reveal promising effects of lycopene in alleviating signs and symptoms of OSF, OLP, and leukoplakia. However, owing to the observed heterogeneity and short follow-up periods, further well-designed studies with long-term therapy and follow-up are highly recommended.

Impact of Intermittent Fasting on Metabolic Syndrome and Periodontal Disease-A Suggested Preventive Strategy to Reduce the Public Health Burden.

Metabolic syndrome (MetS) prevalence continues to climb significantly worldwide in today's ad libitum society. MetS has tremendous societal and economic ramifications, making it imperative to develop effective strategies for preventing and controlling it to alleviate this growing burden. Periodontal disease and MetS are associated with several risk factors. Studies in the past have demonstrated that obesity, cardiovascular illness, and type 2 diabetes mellitus have a negative effect on the severity of the periodontal disease. Patients with metabolic syndrome have elevated serum levels of proinflammatory mediators such as tumor necrosis factor-alpha interleukin-6 and C-reactive protein. Similar inflammatory mediators, such as interleukin-6, tumor necrosis factor-alpha, and C-reactive protein, are increased in patients with severe periodontal disease. Remarkably, intermittent fasting is underpinned by scientific evidence, claiming to be the most effective non-pharmacological, potential therapeutic alternative for combating a wide range of metabolic, inflammatory, and lifestyle-related diseases. Nonetheless, an insufficient investigation has been performed to determine whether intermittent fasting has therapeutic benefits on periodontal inflammation and diseases. Here, we show the interrelationship between metabolic syndrome and periodontal disease and contextualize the beneficial impact of intermittent fasting in modulating the chronic metabolic and periodontal inflammatory response. We also anticipate that this review paves the way for further exploration of intermittent fasting as a unique research paradigm representing a cost-effective alternative strategy to conventional disease management in patients with periodontal diseases and metabolic syndrome which may serve as the foundation for an integrative vision relevant to primary, diagnostic, and therapeutic purposes.

Measuring Plasma Membrane Recycling Using Microscopic and Biochemical Approaches.

The endocytic pathway has an intricate network of vesicular compartments carrying a variety of proteins referred to as cargoes. Endosomal trafficking is exclusively required to transport these cargoes through various intracellular routes for their delivery to the site of action. Among these, recycling of cargoes to the plasma membrane is a crucial pathway for the efficient functioning of the cell. Hence, endosomal cargo recycling assays are crucial to gain insight into the molecular mechanism governing recycling of the cargoes and in turn to understand their key role in maintaining cellular physiology. These assays have been efficiently utilized to study the recycling of adhesion molecules, transporters, channels, receptors, and so on to the plasma membrane. The basic methodology involves labelling of the cargo at the surface, allowing its internalization followed by direct or indirect measurement of the amount of the cargo recycled back to the plasma membrane. These microscopy-based and biochemical methods can be used as a tool to study the role of various trafficking or signaling molecules on the cell surface involved with the recycling of the membrane proteins, by altering their expression either by silencing or overexpressing the gene.

Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

The role of miR-128 in cancer development, prevention, drug resistance, and immunotherapy.

A growing body of evidence has revealed that microRNA (miRNA) expression is dysregulated in cancer, and they can act as either oncogenes or suppressors under certain conditions. Furthermore, some studies have discovered that miRNAs play a role in cancer cell drug resistance by targeting drug-resistance-related genes or influencing genes involved in cell proliferation, cell cycle, and apoptosis. In this regard, the abnormal expression of miRNA-128 (miR-128) has been found in various human malignancies, and its verified target genes are essential in cancer-related processes, including apoptosis, cell propagation, and differentiation. This review will discuss the functions and processes of miR-128 in multiple cancer types. Furthermore, the possible involvement of miR-128 in cancer drug resistance and tumor immunotherapeutic will be addressed.

A Meta-Analysis in Assessing Oxidative Stress Using Malondialdehyde in Oral Submucous Fibrosis.

OBJECTIVE: This study aimed to evaluate the oxidative stress (OS) marker Malondialdehyde (MDA) in oral submucous fibrosis with available literature. MATERIALS AND METHODS: We conducted a literature search electronically in PubMed (MeSH), Science Direct, Scopus, and Google Scholar using specific keywords. RESULTS: A systematic search in PubMed, Science Direct, and Google Scholar identified 334 articles. Of these, four were duplicate reports, and three were animal studies. After reading the abstracts of the collected articles, 288 articles were excluded for the following reasons: low quality, not relevant to the research question, or did not meet the inclusion criteria. The remaining 46 articles were chosen for full-text assessment. Finally, the present qualitative synthesis included 23 articles for evaluation. The selected studies in MDA analysis in a random-effects model showed higher heterogeneity (Q = 477.636, p < 0.001, I2 = 95.394%). The standard difference in mean MDA concentration between oral submucous fibrosis (OSMF) and healthy subjects was estimated as 2.73 nmol/mL (95% confidence interval: 2.08-3.38). CONCLUSION: The selected studies showed significantly higher MDA levels in various biological samples of patients with OSMF. Therefore, further studies are needed to estimate oxidative stress levels by using different biomarkers in OSMF to direct future therapy.

Tinospora cordifolia (Thunb.) Miers (Giloy) inhibits oral cancer cells in a dose-dependent manner by inducing apoptosis and attenuating epithelial-mesenchymal transition.

BACKGROUND: Tinospora cordifolia (Thunb.) Miers (Giloy) has been applied successfully as an anti-inflammatory, anti-diabetic, and even as an anti-cancer agent. Yet, to date, the application of Giloy has not been explored concerning oral cancer. OBJECTIVES: To assess the effect of T cordifolia (Thunb.) Miers (Giloy) extract (TcE) on an oral cancer cell line. METHODS: AW13516 (oral cancer cell line) cells were treated with the prepared aqueous extract of TcE for 24 h at various concentrations ranging between 5 µg/ml and 100 µg/ml and compared with control (cells without treatment). Thee effect of the extracts on apoptosis was assessed by through Annexin V flow cytometry assay and Luminometry based assessment of Caspase 8, 9 and caspase 3/7 activity. RNA was isolated from treated cells and gene expression of selected metastatic genes (MMP1, MMP10, and CXCL8); epithelial-mesenchymal stem cell genes (TWIST1, SNAIL, ZEB1, Oct4) and stemness related genses (Nanog, Sox2) were analyzed by using a quantitative real-time PCR system. The experiments were performed in triplicates. RESULTS: Aqueous extract of TcE was found to induce apoptosis inducer in AW13516 cells in a concentration-dependent manner and was potent even at a low concentration of 5 µg/ml. The apoptosis induction was confirmed with the caspase activity assay. Treatment of the cells with the extract for 24 h exhibited a significant decrease in the expression of EMT genes in a dose-dependent manner without an effect on the metastatic genes. CONCLUSION: Aqueous extract of TcE induces apoptosis-mediated cell death in the oral cancer cell line AW13516 while attenuating its potential for epithelial mesenchymal transition.

Impact of calorie restriction and intermittent fasting on periodontal health.

The scientific evidence indicates that calorie restriction and intermittent fasting are among the appropriate strategies targeting factual causative factors of various inflammatory and lifestyle-related disorders. Periodontitis is a common oral inflammatory disease leading to bone loss that is associated with various systemic problems. Previous studies suggest that calorie restriction may dampen inflammation and concomitant tissue damage under inflammatory conditions, such as periodontal diseases in nonhuman primates. However, insufficient research has been carried out to assess the effects of a calorie-restricted diet on the initiation and progression of periodontal disease in humans. This review of the literature aims to describe the general concepts of calorie restriction, its clinical implications, and related therapeutic potential in controlling periodontal inflammation. The review shows that fasting regimen groups have shown lesser bone loss because of an increase in osteoprogenitor cells than non-fasting groups. Calorie restriction dampens the inflammatory response and reduces circulating inflammatory mediators like tumor necrosis factor-alpha, interleukin-6, matrix metalloproteinase-8, matrix metalloproteinase-9, and interleukin-1-beta in gingival crevicular fluid. However, the incorporation of this form of dietary intervention continues to be challenging in our current society, in which obesity is a major public concern. Calorie restriction and intermittent fasting can play a key role in the cost-effective resolution of periodontal inflammation as a primary prevention strategy for the management of chronic inflammatory diseases, including periodontal diseases.

Proliferative Verrucous Leukoplakia Revisited: A Retrospective Clinicopathological Study.

(1) Objective: To review the criteria proposed by Cerero-Lapiedra et al. and to retrospectively identify the under-diagnosed disease in patients diagnosed with proliferative verrucous leukoplakia. (2) Materials and methods: In this study, we included patients who were diagnosed with leukoplakia (histological label consistent with the clinical diagnosis, n = 95), and cases with a final diagnosis within the spectrum of proliferative verrucous leukoplakia (n = 110) as defined by Batsakis et al. We applied the criteria proposed by Cerero-Lepiedra et al. to screen for the possible cases of proliferative verrucous leukoplakia. (3) Results: Although many of our patients satisfied specific isolated criteria, only 11 cases satisfied specific combinations of the guidelines to satisfy a diagnosis of proliferative verrucous leukoplakia. However, due to the lack of follow-up data, the disease is not confirmed in these 11 cases. (4) Conclusion: A limited number of cases of proliferative verrucous leukoplakia were diagnosed using the criteria given by Cerero-Lapiedra et al. The true natural history of the disease could not be studied due to the lack of follow-up data. (5) Clinical relevance: Proliferative verrucous leukoplakia presenting as hyperkeratosis or mild epithelial dysplasia are often not followed up, and they subsequently transform into carcinoma. Thus, clinicians must be vigilant whenever they encounter leukoplakia, especially with multifocal presentations. In such cases, the follow-up data are the key to understanding the true nature of the disease entity.

Zinc supplementation for prevention and management of recurrent aphthous stomatitis: a systematic review.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is a very common oral ulcerative disease with no definitive cure. Growing evidence suggests a significant association between zinc deficiency and RAS. OBJECTIVES: To assess the efficacy of systemic zinc supplementations in the prevention and management of RAS. METHODS: Five databases were searched for all English and Chinese studies published up to November 2020. All clinical trials that assessed the efficacy of zinc supplementations in the management and/or prevention of RAS were included. RESULTS: Seven clinical trials comprising 482 RAS patients (250 in zinc group) fulfilled the inclusion criteria. The follow-up period ranged from three months to one year. Five studies showed significantly better efficacy of zinc in reducing the recurrence rates of RAS, whereas two studies did not report any significant differences compared to the controls. Four studies reported on signs/symptoms of RAS, three of which showed superior outcomes in favour of zinc, while one study reported comparable results. CONCLUSION: Zinc supplementation seems to be efficacious in the management and prevention of RAS. However, further clinical trials with standardized methodologies and adequate follow-up periods are required to confirm the efficacy of zinc supplementations.

Reply to the letter regarding "Chronic mechanical irritation and oral squamous cell carcinoma: A systematic review and meta-analysis".

Dear Editor, We thank Dr. Jian Xie for the valuable inputs on our paper titled 'Chronic mechanical irritation and oral squamous cell carcinoma: A systematic review and meta-analysis [1].' The first concern of Dr. Xie was that we had included two studies that were based on the same population. We re-examined these papers, one was published in 2010 [2] and the other in 2017 [3] by the same group of authors. Given the significant time difference between the two papers, we did not want to presume they were from the same sample population. There is no clear evidence that they are from the same sample population. Read more in PDF.

Chronic mechanical irritation and oral squamous cell carcinoma: A systematic review and meta-analysis.

The objective of the present article was to qualitatively and quantitatively review the association between chronic mechanical irritation and oral squamous cell carcinoma (OSCC). PubMed, SCOPUS, and Web of Science databases were searched using the keyword combinations "chronic trauma and oral squamous cell carcinoma; chronic irritation and oral squamous cell carcinoma; chronic irritation and oral cancer; and chronic trauma and oral cancer." Duplicates and irrelevant articles were excluded after the title and abstract screening. The full texts of the remaining articles were assessed using selection criteria. A total of 375 (PubMed-126; SCOPUS-152; WOS-97) articles were screened, and 343 duplicates and irrelevant articles were excluded from the study. Only 9 of the remaining 32 articles met the selection criteria and were included in the qualitative analysis. Buccal mucosa and tongue, being highly prone to chronic irritation through the dental prosthesis, were the common sites for OSCC. Edentulous subjects with ill-fitting dentures were at a high risk of developing chronic irritation associated-OSCC. According to the Joanna Briggs Institute of risk assessment, eight of the nine included studies had a low risk of bias. The quantitative analysis showed a significant association (p < 0.00001) between the chronic oral mucosal irritation and OSCC with an overall risk ratio of 2.56 at a confidence interval of 1.96-3.35. Chronic oral mucosa irritation has a significant association with OSCC, and the nature of association could be that of a potential co-factor (dependent risk factor) rather than an independent risk factor.

Assessing the potential use of chitosan scaffolds for the sustained localized delivery of vitamin D.

Vitamin D is a commonly used bone modulator in regenerative medicine. Several modalities have been explored for the delivery of vitamin D including nanoparticles and scaffold. The present study aimed to assess the potential use of a bio-degradable chitosan scaffold for the delivery of vitamin D. The objectives included fabrication of a bio-degradable chitosan scaffold, integration of vitamin D into the scaffold, characterization of the vitamin D integrated scaffold. Characterization was carried out using, X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The structure of the scaffold was assessed by scanning electron microscopy. The scaffold was placed in phosphate buffer saline and the release duration of vitamin D was observed using UV spectrophotometry. Dental pulp mesenchymal stem cells were added to the scaffold to study the scaffold associated toxicity and the functionality of the scaffold released vitamin D. The vitamin D release period from the scaffold was estimated to be for 80 hrs. MTT assay of the stem cells was comparable to that of the control group (stem cells cultured in media) inferring that the scaffold is not toxic towards the stem cells. The positive alizarin red S staining, a higher expression of alkaline phosphatase, osteocalcin, and RunX2 confirmed the functional capability (osteogenic differentiation of the stem cells) of the released vitamin D. Based on the data from the present study, it can be inferred that chitosan scaffold can be used for the sustained delivery of functional vitamin D for 3-5 days.

Protein-templated gold nanoclusters as specific bio-imaging probes for the detection of Hg(ii) ions in in vivo and in vitro systems: discriminating between MDA-MB-231 and MCF10A cells.

Gold nanoclusters (AuNCs) synthesized within a protein (Human Serum Albumin, HSA) template exhibited intense red luminescence accompanied by a quantum yield >10% and remarkable photo and cluster-core stability for a prolonged period (more than a year). These photoluminescent nanoclusters (NCs) were resistant to chemical and thermal perturbations but break down selectively and highly sensitively in the presence of mercury, Hg(ii), ions. The AuNCs were efficient in quantifying Hg(ii) ions in solution as well as bound to the hormone insulin. By exploiting the auto-fluorescence of these AuNCs, we demonstrated that our AuNCs were able to sense Hg(ii) ions at single-molecule sensitivity using Fluorescence Correlation Spectroscopy (FCS), highlighting a detection limit in the sub-nanomolar regime. The translational diffusion time of the AuNCs decreased significantly upon the interaction with Hg(ii) ions and resulted in the formation of smaller sized clusters. A cell viability study reveals the non-toxic nature of these nano-probes, which thus can be used for cell imaging. Interestingly, a cell line-based study reveals that the fluorescence intensity of AuNCs could be detected in cancerous MDA-MB-231 cells but not in non-cancerous breast-derived MCF10A cells. Further, time lapse fixed cell imaging by confocal microscopy revealed that the fluorescence of AuNCs could be quenched by Hg(ii) ions inside the MDA-MB-231 cells. Thus the objective of our study is to appraise the sensitive in vivo as well as in vitro detection of Hg(ii) ions using AuNCs as a probe.

EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation.

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.

SNX27-retromer assembly recycles MT1-MMP to invadopodia and promotes breast cancer metastasis.

A variety of metastatic cancer cells use actin-rich membrane protrusions, known as invadopodia, for efficient ECM degradation, which involves trafficking of proteases from intracellular compartments to these structures. Here, we demonstrate that in the metastatic breast cancer cell line MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix metalloprotease, MT1-MMP. We further found that MT2-MMP, another abundantly expressed metalloprotease, is also invadopodia associated. MT1- and MT2-MMP showed a high degree of colocalization but were located on the distinct endosomal domains. Retromer and its associated sorting nexin, SNX27, phenocopied each other in matrix degradation via selectively recycling MT1-MMP but not MT2-MMP. ITC-based studies revealed that both SNX27 and retromer could directly interact with MT1-MMP. Analysis from a publicly available database showed SNX27 to be overexpressed or frequently altered in the patients having invasive breast cancer. In xenograft-based studies, SNX27-depleted cell lines showed prolonged survival of SCID mice, suggesting a possible implication for overexpression of the sorting nexin in tumor samples.

Essential and selective role of SNX12 in transport of endocytic and retrograde cargo.

The endosomal protein-sorting machineries play vital roles in diverse physiologically important cellular processes. Much of the core membrane-sorting apparatus is conserved in evolution, such as retromer, which is involved in the recycling of a diverse set of cargoes via the retrograde trafficking route. Here, in an RNAi-based loss-of-function study, we identified that suppression of SNX12 leads to a severe blockage in CIM6PR (also known as IGF2R) transport and alters the morphology of the endocytic compartments. We demonstrate that SNX12 is involved in the early phase of CIM6PR transport, and mediates receptor recycling upstream of the other well-established SNX components of retromer. Ultra-structural analysis revealed that SNX12 resides on tubulo-vesicular structures, despite it lacking a BAR domain. Furthermore, we illustrate that SNX12 plays a key role in intraluminal vesicle formation and in the maturation of a subpopulation of early endosomes into late endosomes, thereby regulating selective endocytic transport of cargo for degradation. This study therefore provides evidence for the existence of early endosomal subpopulations that have differential roles in the sorting of the cargoes along endocytic degradative pathways.