Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer.

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.

Ultrahigh Photo-Responsivity and Detectivity in 2D Bismuth Sulfide Photodetector for Vis-NIR Radiation.

Bismuth sulfide (Bi2S3) exhibits a direct energy bandgap and an exceptional optical absorption capability over a broadband radiation, thus presents a novel class of 2D photodetector material. The field effect transistor (FET) photodetector device is fabricated from 2D Bi2S3. An anomalous variation in the transport characteristics of 2D Bi2S3 is observed with the variation in temperature. The electrical resistance reduces by 99.26% at 10 K compared to the response at 300 K. Defects due to the bismuth and sulfur vacancies play a critical role in the dramatic behavior, which is confirmed using photoluminescence, time-resolved photoluminescence, Hall measurements, and energy dispersive X-ray spectroscopy. The density functional theory calculations provide a significant insight into the thermodynamic properties of intrinsic defects in Bi2S3. Moreover, the effect of gate bias on responsivity additionally confirms its invariance at low temperature. The Bi2S3 based FET photodetector achieves ultrahigh responsivity in the order of ≈106 A W-1 and detectivity of ≈1014 Jones. Moreover, the external quantum efficiency of ≈107% is measured in a wide spectrum of optical illumination (532 to 1064 nm) with a noise-equivalent power of 3.5 × 10-18 W/√Hz at a bias of 0.2 V. The extraordinary performance of Bi2S3 photodetector outstands 2D photodetectors.

A comprehensive review on spatial and temporal variation of arsenic contamination in Ghaghara basin and its relation to probable incremental life time cancer risk in the local population.

Spatial and temporal variations have been found in the levels of arsenic (As) throughout the groundwater of the Ghaghara basin. Fifteen out of twenty-five districts in this basin are reported to be affected by As, where the levels of As in groundwater and soil exceed the permissible limits set by the WHO (10 µgl-1) and FAO (20 mgkg-1) respectively. These districts include a total of four municipalities in Nepal and eighty-six blocks in India, all of which have varying degrees of As contamination. Approximately 17 million people are at risk of As poisoning, with more than two orders of magnitude higher potential lifetime incremental cancer risk, constituting over 153 thousand potential additional cases of cancer due to As-contaminated drinking water. Out of the 90 As-contaminated blocks in the Ghaghara basin, 4 blocks have about 7-fold higher potential risk of developing cancer, 49 blocks have 8-37-fold higher risk, and 37 blocks have up to 375-fold higher risk compared to the upper limit of the USEPA acceptable range, which is 1 × 10-6-1 × 10-4. High accumulation of As has been reported in the nails, hair, and urine of local inhabitants, with higher levels observed in females than males. The toxicity of As is manifested in terms of a higher occurrence of various diseases. Reproductive endpoints, such as increased incidences of preterm birth, spontaneous abortion, stillbirth, low-birth weight, and neonatal death, have also been reported in the basin. The level of As in tube wells has been found to be negatively correlated with the depth (r = -0.906), and tube wells with high levels of As (>150 µgl-1) are generally located within close proximity (<10 km) to abandoned or present meander channels in the floodplain areas of the Ghaghara river. In addition to As contamination, the water quality index (WQI) in the Ghaghara basin is poor according to the BIS standards for drinking water. Groundwater in six out of fifteen districts is unsuitable for drinking purposes, with a WQI exceeding 100. The levels of As in agricultural soil in many villages of Ballia, Bahraich, and Lakhimpur Kheri districts have exceeded the FAO limit. Water from deep tube wells has been found to be relatively safe in terms of As content, and thus can be recommended for drinking purposes. However, the use of surface water needs to be encouraged for irrigation purposes in order to preserve soil health and reduce As contamination in the food chain, thereby minimizing the risk of cancer.

Diagnostic Dilemma of Vanishing Bone Disease - A Case Report and Review of Literature.

Rationale: Vanishing bone disease (VBD) is a rare bone disorder in which progressive osteolysis may lead to complete disappearance of involved bones. The diagnosis of this disease requires a high degree of clinical suspicion. We present a case of progressive osteolysis of mandible in a patient. Patient Concerns: The patient had been without definitive diagnosis and treatment for over a year. Diagnosis: Diagnosis was made by exclusion of genetic, traumatic, inflammatory, infective, endocrine and neoplastic aetiologies and by carefully correlating clinical, imaging and histopathological findings of the patient. Treatment: Segmental resection of the advancing edge of the lesion was carried out. Outcome: The patient is disease free, with no evidence of further osteolysis, after six months of follow-up. Take-Away Lessons: This article describes the exclusion-based approach adopted to diagnose a case of VBD, aiming to standardise a workup for the diagnosis.

Alteration of oral bacteriome of smokeless tobacco users and their association with oral cancer.

Smokeless tobacco (SLT) is certainly one of the major risk factors associated with oral cancer. Disruption of oral microbiota-host homeostasis contributes to the progression of oral cancer. Here, we profiled SLT users' oral bacterial composition and inferred their functions by sequencing 16S rDNA V3-V4 region and PICRUSt2, respectively. Oral bacteriome of SLT users (with or without oral premalignant lesions), SLT with alcohol co-users, and non-SLT consumers were compared. Oral bacteriome is shaped primarily by SLT use and the incidence of oral premalignant lesions (OPL). A significantly increased bacterial α-diversity was monitored in SLT users with OPL compared to in SLT users without OPL and non-users, whereas ß-diversity was significantly explained by OPL status. Overrepresented genera were Prevotella, Fusobacterium, Veillonella, Haemophilus, Capnocytophaga, and Leptotrichia in SLT users having OPL. LEfSe analysis identified 16 genera as a biomarker that were differentially abundant in SLT users having OPL. The functional prediction of genes significantly increased for several metabolic pathways, more importantly, were nitrogen metabolism, nucleotide metabolism, energy metabolism, and biosynthesis/biodegradation of secondary metabolites in SLT users having OPL. Furthermore, HPV-16 and EBV, but not HPV-18, were considerably connected with the SLT users having OPL. Overall, this study provides evidence that SLT utilization and OPL development are associated with oral bacteriome dysbiosis indicating the enrichment of bacterial species known for their contribution to oral carcinogenesis. Therefore, delineating the cancer-inducing bacterial population in SLT users will facilitate the future development of microbiome-targeted therapies. KEY POINTS: • SLT consumption significantly elevates oral bacterial diversity. • Prevalent significant genera are Prevotella, Veillonella, and Haemophilus in SLT users with OPL. • SLT promotes the occurrence of the cancer-inducing bacterial population.

Exploration of a Large Virtual Chemical Space: Identification of Potent Inhibitors of Lactate Dehydrogenase-A against Pancreatic Cancer.

It is imperative to explore the gigantic available chemical space to identify new scaffolds for drug lead discovery. Identifying potent hits from virtual screening of large chemical databases is challenging and computationally demanding. Rather than the traditional two-dimensional (2D)/three-dimensional (3D) approaches on smaller chemical libraries of a few hundred thousand compounds, we screened a ZINC library of 15 million compounds using multiple computational methods. Here, we present the successful application of a virtual screening methodology that identifies several chemotypes as starting hits against lactate dehydrogenase-A (LDHA). From 29 compounds identified from virtual screening, 17 (58%) showed IC50 values < 63 µM, two showed single-digit micromolar inhibition, and the most potent hit compound had IC50 down to 117 nM. We enriched the database and employed an ensemble approach by combining 2D fingerprint similarity searches, pharmacophore modeling, molecular docking, and molecular dynamics. WaterMap calculations were carried out to explore the thermodynamics of surface water molecules and gain insights into the LDHA binding pocket. The present work has led to the discovery of two new chemical classes, including compounds with a succinic acid monoamide moiety or a hydroxy pyrimidinone ring system. Selected hits block lactate production in cells and inhibit pancreatic cancer cell lines with cytotoxicity IC50 down to 12.26 µM against MIAPaCa-2 cells and 14.64 µM against PANC-1, which, under normoxic conditions, is already comparable or more potent than most currently available known LDHA inhibitors.

An interesting case of systemic lupus erythematosus with multiple myeloma.

A rare association exists between systemic lupus erythematosus (SLE) and multiple myeloma (MM). SLE is associated with a variety of symptoms. A combination of MM and SLE is uncommon in the young population. An unusual case of SLE associated with MM is described here. We present the case of a 39-year-old woman who was a known case of SLE and presented with severe chest and abdominal pain. We summarize the clinical characteristics of MM in SLE. The possible mechanisms that could be at the root of this association are also discussed.

An Audit of Diagnostic Disparity between Intraoperative Frozen Section Diagnosis and Final Histopathological Diagnosis of Central Nervous System Lesions at a Tertiary Care Center.

Introduction Evaluation of intraoperative squash smear and frozen section (FS) in central nervous system (CNS) neoplasms is consistently practiced for rapid assessment and has several advantages to its credence. It is an invaluable tool to ensure adequacy of tissue obtained to establish the diagnosis. Moreover, it aids in guiding the surgeon for critical decisions regarding the extent of resection. Although molecular markers have been integrated with morphology in the revised 2016 World Health Organization classification of brain tumors, precise morphological assessment still remains the foundation for the diagnosis and rapid intraoperative assessment of morphological details is equally critical and rewarding. Objective This study aims to audit the diagnostic disparity between intraoperative diagnoses based on a combination of squash cytology and FS in cases of CNS lesions with gold standard, final diagnosis based on examination of formalin fixed paraffin embedded hematoxylin, and eosin-stained tissue sections. Materials and Methods All intraoperative squash cytology and FS reported for CNS lesions from January 2017 to December 2020 were reviewed. The cases were categorized into three groups-group 1: when diagnosis of intraoperative diagnosis based on a combination of squash cytology and FS was same as the final histopathological diagnosis (concordant), group 2: partially concordant, and group 3: discordant cases. Statistical Analysis Descriptive statistics was used to classify the data and diagnostic accuracy was calculated. Results Complete concordance was present in 69.96% (191/273) cases, 20.1% (55/273) cases showed partial concordance, and 9.89% (27/273) cases were discordant with histopathological diagnosis. Out of the 27 discordant cases, misclassification of tumor type was the most common category (11 cases, 40%), followed by grading mismatch (7 cases, 25.9%), and misdiagnosis of tumor versus nontumor conditions (9 cases, 33.3%). Conclusion Our study shows that combination of intraoperative squash cytology and FS shows a high percentage of accuracy in arriving at intraoperative diagnosis in cases of intracranial lesions. Regular audits of discordant cases should be conducted by surgeons and pathologists as part of a quality assurance measure to sensitize themselves with the potential pitfalls, minimizing misinterpretation and helping in providing a more conclusive opinion to the operating surgeons.

Attention Detection by Heartbeat and Respiratory Features from Radio-Frequency Sensor.

This work presents a study on users' attention detection with reference to a relaxed inattentive state using an over-the-clothes radio-frequency (RF) sensor. This sensor couples strongly to the internal heart, lung, and diaphragm motion based on the RF near-field coherent sensing principle, without requiring a tension chest belt or skin-contact electrocardiogram. We use cardiac and respiratory features to distinguish attention-engaging vigilance tasks from a relaxed, inattentive baseline state. We demonstrate high-quality vitals from the RF sensor compared to the reference electrocardiogram and respiratory tension belts, as well as similar performance for attention detection, while improving user comfort. Furthermore, we observed a higher vigilance-attention detection accuracy using respiratory features rather than heartbeat features. A high influence of the user's baseline emotional and arousal levels on the learning model was noted; thus, individual models with personalized prediction were designed for the 20 participants, leading to an average accuracy of 83.2% over unseen test data with a high sensitivity and specificity of 85.0% and 79.8%, respectively.

To evaluate the functional and anatomical outcomes for autologous retinal autograft with Finesse™ Flex Loop for failed macular holes.

Purpose: To evaluate the functional and anatomical outcomes for autologous retinal autograft with Finesse™ Flex Loop for failed macular holes. Methods: This is a retrospective study analyzing medical records of consecutive patients with refractory macular hole (at least 1 prior surgery) and eyes with retinal detachment with coexisting macular holes (MH). Optical coherence tomography (OCT) of the macula was performed before and after surgery. The primary study outcome evaluated were the functional and anatomic macular hole closure, and secondary outcomes were improvement in visual acuity and restoration of the outer retinal bands, external limiting membrane, and ellipsoid zone in eyes with acquisition of autologous retinal transplant using Finesse Loop. Results: The study included eight eyes of eight patients; retinal autograft was performed in six (75.0%) and autologous retinal transplantation (ART) with rhegmatogenous retinal detachment (RRD) was performed in two (25.0%) eyes. The average MH basal diameter in the study was 1310.88 ± 138.63 µm. The successful hole closure rate was observed to be 75% (6 eyes) and 100% retinal reattachment was observed in ART with RRD. Statistically significant (P = 0.001) improvement was noted for preop and postop visual acuity gain for ART acquisition and postop macular hole closure. The restoration of the external limiting membrane (ELM) and ellipsoid zone (EZ) was observed in 37.5% (3 eyes) of patients. Conclusion: Finesse™ Flex Loop can be used to harvest retinal tissue and it provides good anatomical and functional outcomes for failed macular hole.

Smokeless tobacco consumption induces dysbiosis of oral mycobiome: a pilot study.

Smokeless tobacco (SLT) alters the oral microbiome of smokeless tobacco users. Dysbiosis of oral bacteriome has been determined; however, the mycobiome of SLT users has not been characterized. The oral mycobiome was assayed by amplification and sequencing of the fungal internal transcribed spacer (ITS1) region from oral swab samples of non-SLT users, SLT users (with or without oral lesions), and SLT with alcohol users. We observed that the richness and diversity of oral mycobiome were significantly decreased in SLT with oral lesions users than in non-users. The ß-diversity analysis showed significant dissimilarity of oral mycobiome between non-users and SLT with oral lesions users. Linear discriminant analysis effect size and random forest analysis of oral mycobiome affirm that the genus Pichia was typical for SLT with oral lesions users. Prevalence of the fungal genus Pichia correlates positively with Starmerella, Mortierella, Fusarium, Calonectria, and Madurella, but is negatively correlated with Pyrenochaeta, Botryosporium, and Alternaria. Further, the determination of oral mycobiome functionality showed a high abundance of pathotroph-saprotroph-symbiotroph and animal pathogen-endophyte-epiphyte-undefined saprotroph at trophic and guild levels, respectively, indicating possibly major changes in normal growth repression of types of fungi. The oral mycobiome in SLT users was identified and comprehensively analyzed for the first time. SLT intake is associated with oral mycobiome dysbiosis and such alterations of the oral mycobiome may contribute to oral carcinogenesis in SLT users. This study will provide a basis for further large-scale investigations on the potential role of the mycobiome in SLT-induced oral cancer. KEY POINTS: • SLT induces dysbiosis of the oral microbiome that can contribute to oral cancer. • Oral mycobiome diversity is noticeably reduced in SLT users having oral lesions. • Occurrence of Pichia can be used as a biomarker for SLT users having oral lesions.

Comparison of patient satisfaction with red-free (green) versus yellow light using binocular indirect ophthalmoscope for retinal examination.

Purpose: Comparison of patient satisfaction with red-free (green) versus yellow light using binocular indirect ophthalmoscope for retinal examination. Methods: This is an observational questionnaire-based study of 100 myopes in the age group of 18-40 years coming for a routine check-up or for refractive surgery workup. The examination was done using an indirect ophthalmoscope and a 20D lens with green or yellow light and was assigned in two groups randomly using the coin toss method, following which, a questionnaire was used to assess the following parameters: a) level of comfort, b) any complaints of discomfort during examination, d) preference of the used light source in future, e) grading of discomfort on a linear scale, and f) patient cooperation and duration of examination. Results: Patients were randomized for observation with IDO using either green light (n = 55) or yellow light (n = 45) filter. In the study, 46 patients (83.6%) were very comfortable and only 9 patients (16.4%) experienced mild discomfort when using red-free (green) light, while only 3 patients (6.7%) were very comfortable and 31 (68.9%) had mild discomfort when using yellow light. The complaints of watering with yellow and green light were noted in 36 patients (80.0%) and 15 patients (27.3%), pain in 13 patients (28.9%) and 3 patients (5.5%), light sensitivity in 29 patients (64.4%) and 4 patients (7.3%), respectively, all being significantly more in yellow light category (P < 0.001). The time of examination was significantly more in yellow light category with 83 ± 10.75 seconds (P < 0.001). Conclusion: Retinal examination using the green filter of indirect ophthalmoscope is more comfortable in examining the patients as compared to routine yellow light with decreased number of complaints, lesser examination time, and better patient cooperation.

Perceptions of Good Health and Impact of COVID-19 Among Adolescents in a Low-Income Urban Agglomerate in Delhi, India: A Qualitative Study.

Adolescents constitute 16% of the global population and are susceptible to adverse health and illness from substance abuse, unhealthy diet, physical inactivity, and high-risk sexual behaviors. We conducted this study to assess the perceptions of good health, health-seeking behavior, and health service utilization among adolescents living in a low-income urban neighborhood after the second wave of the COVID-19 pandemic. A total of 23 adolescents, including 12 males and 11 females, were interviewed. Adolescents' perceived body image and size considerations apart from functioning at an optimum physical capacity as the principal attributes of good health, which was possible through the intake of a healthy diet and exercise. Adolescents were likely to be aware of the addiction potential and risk of cancer from using tobacco and alcohol, but attitudes towards eschewing their use were ambivalent. Adolescents perceived themselves as lacking access to reliable, adequate, and validated sources of sexual and reproductive health information. Knowledge and utilization of adolescent health services in this area were negligible, suggestive of the need to strengthen these services and improve the program outreach.

Spoligotyping of Mycobacterium tuberculosis isolates from Pulmonary Tuberculosis patients from North Karnataka, India.

Tuberculosis (TB) is a leading cause of morbidity and mortality in low income countries. Multi-drug resistant (MDR-TB) is seen as the reason for many TB outbreaks globally and is also a threat to control programmes. India accounts for 27% TB cases worldwide. Our study was undertaken to understand the outbreaks related to MTB. All the sputum samples were subjected to microscopy and smear positive samples were cultured on Lowenstein-Jensen (L-J) media. Identification was carried by biochemical analysis. A total of 57 isolates were subjected to Drug Susceptibility testing (DST) and spoligotyping, where eleven MDR-TB isolates were confirmed, of which ten were SIT1/Beijing and one SIT53/T1. Spoligotyping results showed that the predominant lineage in this region was SIT1/Beijing followed by SIT124/U and the strains which did not match spoligodatabase were named as orphans. In this study, MDR-TB was associated with SIT1/Beijing and mono resistance belonged to CAS1_DEL.

Primary retroperitoneal extraovarian granulosa cell tumor.

Extraovarian granulosa cell tumors (GCTs) develop from ectopic gonadal tissue situated along the embryonal route of the genital ridge. Primary retroperitoneal tumors are extremely rare, with an incidence of 02% -06% and 80-85% probability of malignancy. Only eight such case reports have been published previously. We herein, report a rare case of extraovarian retroperitoneal GCT in a 55-year-old woman who presented with intermittent left lumbar region pain of one-year duration. She had a history of hysterectomy and bilateral salpingo-oophorectomy 8 years ago for uterine leiomyoma. Laparotomy revealed a retroperitoneal mass measuring 8cm x 10cm x 20cm in size, solid cystic with areas of necrosis and hemorrhage. The gross features, classical histopathology, and positive immunostaining of the retroperitoneal mass with inhibin, calretinin, PR, WT1 and immunonegativity for EMA were characteristic of adult-type GCT. Excluding any previous history of primary ovarian GCT in this patient, a de-novo retroperitoneal diagnosis was established.

Development of Transition-Metal-Free Lewis Acid-Initiated Double Arylation of Aldehyde: A Facile Approach Towards the Total Synthesis of Anti-Breast-Cancer Agent.

This work describes a mild and robust double hydroarylation strategy for the synthesis of symmetrical /unsymmetrical diaryl- and triarylmethanes in excellent yields using Lambert salt (0.2-1.0 mol%). Despite the anticipated challenges associated with controlling selective product formation, unsymmetrical diaryl- and triarylmethanes products are obtained unprecedentedly. A highly efficient gram scale reaction has also been reported (TON for symmetrical product=475 and for unsymmetrical product=390). The synthetic utility of the methodology is demonstrated by the preparation of several unexplored diaryl- and triarylmethane-based biologically relevant molecules, such as arundine, vibrindole A, turbomycin B, and certain anti-inflammatory agents. A total synthesis of an anti-breast-cancer agent is also demonstrated. Control experiments, Hammett analysis, HRMS and GC-MS studies reveal the reaction intermediates and reaction mechanism.

Primary retroperitoneal extraovarian granulosa cell tumor

Extraovarian granulosa cell tumors (GCTs) develop from ectopic gonadal tissue situated along the embryonal route of the genital ridge. Primary retroperitoneal tumors are extremely rare, with an incidence of 02% -06% and 80-85% probability of malignancy. Only eight such case reports have been published previously. We herein, report a rare case of extraovarian retroperitoneal GCT in a 55-year-old woman who presented with intermittent left lumbar region pain of one-year duration. She had a history of hysterectomy and bilateral salpingo-oophorectomy 8 years ago for uterine leiomyoma. Laparotomy revealed a retroperitoneal mass measuring 8cm x 10cm x 20cm in size, solid cystic with areas of necrosis and hemorrhage. The gross features, classical histopathology, and positive immunostaining of the retroperitoneal mass with inhibin, calretinin, PR, WT1 and immunonegativity for EMA were characteristic of adult-type GCT. Excluding any previous history of primary ovarian GCT in this patient, a de-novo retroperitoneal diagnosis was established.

Prevalence and Social Contextual Factors of Smokeless Tobacco Use: Insights from Schools of Delhi, India.

BACKGROUND: It was observed that adult users start tobacco use in childhood or adolescence. The influence of digital and print media, social acceptance among peers, and in order to mimic role models from films attracts youth towards tobacco. Hence this study was conducted to assess the prevalence of smokeless tobacco (SLT) use among school-going adolescents with the assessment of the influencing factors such as exposure at school, home, and public places along with the role of various media in SLT use by adolescents. METHODS: Cross sectional study was conducted with 860 students of class 9th -12th enrolled in schools. Purposive sampling of three schools was done from the study area to reach desired sample size. All the students of class 9th - 12th within selected schools were given chance to participate in the study. Data collection was done using pretested modified Global Youth Tobacco Survey (GYTS) questionnaire. RESULTS: Consumption of smokeless tobacco (SLT) was observed in 79 (11%) of 714 subjects. Significantly higher proportion (18.4%) of study subjects consumed SLT who were exposed to tobacco at school premises and 19.1% of study subjects who were exposed to teacher using SLT in schools compared to non-exposed group. (p=0.016). It was observed that 8.1% of subjects without any exposure to tobacco at home and 9.8% of subjects without exposure to tobacco at outdoor public space consumed SLT. Consumption of SLT use was highest (16.7%) among subjects exposed to tobacco advertisement or promotion through social media (p=0.04). CONCLUSION: High prevalence of SLT was detected among adolescents in school. Factors such as exposure to tobacco at home, public places, school and school teacher using SLT, exposure of tobacco advertisement and promotion via different modes of media was found to be significantly associated with the use of SLT in the adolescents of school.
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Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum.

BACKGROUND: Plasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT. METHODS: Using the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter's substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron. RESULTS: The combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein. CONCLUSION: The structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.