Carrier Family Research Articles

Dihydroartemisinin inhibits ATP6 activity, reduces energy metabolism of hepatocellular carcinoma cells, promotes apoptosis and inhibits metastasis via CANX.

Dihydroartemisinin (DHA) may inhibit the migration and invasion of liver cancer cells by reducing ATP synthase production (specifically ATP1A1 and ATP5H) through the calcium/calmodulin dependent protein kinase kinase 2/solute carrier family 8 member B1 signaling pathway. However, it is unclear whether DHA regulates ATP synthase activity by modulating other calcium ion signals to inhibit the energy metabolism and the transfer of hepatocellular carcinoma (HCC) cells. Using the Gene Expression Profiling Interactive Analysis database, a search for specific expression genes in liver cancer tissues was performed. Human HCC HuH-7 and Li-7 cells were used to produce CANX overexpression and small interfering RNA cell models. The study assessed changes in cell proliferation, apoptosis, migration and invasion. Reactive oxygen species production, ATP production, mitochondrial membrane potential (JC-1), NAD+/NADH ratio and mitochondrial fluorescence were also evaluated. Western blotting was used to assess changes in CANX, ATP6V1 domain (ATP6V1F) and V0 domain (ATP6V0B) protein expression levels. The results demonstrated that CANX is highly expressed in liver cancer tissues. Furthermore, CANX regulated malignant biological behavior, mediated mitochondrial function and energy metabolism. However, these effects were inhibited by DHA, which decreased the expression of CANX, ATP6V0B and ATP6V1F. The findings of the present study underscore the central role of CANX in affecting the malignant biological behavior of liver cancer cells by regulating mitochondrial function and energy metabolism. Additionally, they indicate that DHA serves an anticancer role by inhibiting CANX expression.

Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells.

Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers. Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up. Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway.

Uncovering the Cardioprotective Potential of Diacerein in Doxorubicin Cardiotoxicity: Mitigating Ferritinophagy-Mediated Ferroptosis via Upregulating NRF2/SLC7A11/GPX4 Axis.

Doxorubicin (DOX)-induced cardiotoxicity (DIC) is a life-threatening clinical issue with limited preventive approaches, posing a substantial challenge to cancer survivors. The anthraquinone diacerein (DCN) exhibits significant anti-inflammatory, anti-proliferative, and antioxidant actions. Its beneficial effects on DIC have yet to be clarified. Therefore, this study investigated DCN's cardioprotective potency and its conceivable molecular targets against DIC. Twenty-eight Wister rats were assigned to CON, DOX, DCN-L/DOX, and DCN-H/DOX groups. Serum cardiac damage indices, iron assay, oxidative stress, inflammation, endoplasmic reticulum (ER) stress, apoptosis, ferritinophagy, and ferroptosis-related biomarkers were estimated. Nuclear factor E2-related factor 2 (NRF2) DNA-binding activity and phospho-p53 immunoreactivity were assessed. DCN administration effectively ameliorated DOX-induced cardiac cytomorphological abnormalities. Additionally, DCN profoundly combated the DOX-induced labile iron pool expansion alongside its consequent lethal lipid peroxide overproduction, whereas it counteracted ferritinophagy and enhanced iron storage. Indeed, DCN valuably reinforced the cardiomyocytes' resistance to ferroptosis, mainly by restoring the NRF2/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling axis. Furthermore, DCN abrogated the cardiac oxidative damage, inflammatory response, ER stress, and cardiomyocyte apoptosis elicited by DOX. In conclusion, for the first time, our findings validated DCN's cardioprotective potency against DIC based on its antioxidant, anti-inflammatory, anti-ferroptotic, and anti-apoptotic imprint, chiefly mediated by the NRF2/SLC7A11/GPX4 axis. Accordingly, DCN could represent a promising therapeutic avenue for patients under DOX-dependent chemotherapy.

Metformin improves nonalcoholic fatty liver disease in db/db mice by inhibiting ferroptosis

Nonalcoholic fatty liver disease (NAFLD) is the primary complication of type 2 diabetes (T2DM)-related liver disease, lacking effective treatment options. Metformin (Met), a widely prescribed anti-hyperglycemic medication, has been found to protect against NAFLD. Ferroptosis, a newly discovered form of cell death, is associated with the development of NAFLD. Despite this association, the extent of Met's protective effects on NAFLD through the modulation of ferroptosis has yet to be thoroughly investigated. In the present study, the administration of erastin or Ras-selective lethal 3 (RSL3), both known ferroptosis inducers, resulted in elevated cell mortality and reduced cell viability in AML12 hepatocytes. Notably, Met treatment demonstrated the capacity to mitigate these effects. Furthermore, we observed increased ferroptosis levels in both AML12 hepatocytes treated with palmitate and oleate (PA/OA) and in the liver tissue of db/db mice. Met treatment demonstrated significant reductions in iron accumulation and lipid-related reactive oxygen species production, simultaneously elevating the glutathione/glutathione disulfide ratio in both PA/OA-treated AML12 hepatocytes and the liver tissue of db/db mice. Interestingly, the anti-ferroptosis effects of Met were significantly reversed with the administration of RSL3, both in vitro and in vivo. Mechanistically, Met treatment regulated the glutathione peroxidase 4/solute carrier family 7 member 11/acyl-CoA synthetase long-chain family member 4 axis to alleviate ferroptosis in NAFLD hepatocytes. Overall, our findings highlight the crucial role of ferroptosis in the development of T2DM-related NAFLD and underscore the potential of Met in modulating key factors associated with ferroptosis in the context of NAFLD.

Luteolin target HSPB1 regulates endothelial cell ferroptosis to protect against radiation vascular injury.

Vascular endothelial damage due to ionizing radiation is the main pathological process of radiation injury and the main cause of damage to various organs in nuclear accidents. Ferroptosis plays an important role in ionizing radiation-induced cell death. We have previously reported that luteolin is highly resistant to ferroptosis. In the present study, body weight, microvessel count, H&E, and Masson staining results showed that luteolin rescued radial vascular injury in vivo. Cell Counting Kit 8 (CCK8), Giemsa staining clarified the anti-ferroptosis ability of luteolin with low toxicity. Malondialdehyde (MDA), superoxide dismutase (SOD), NADP+/NADPH, Fe2+ staining, dihydroethidium (DHE) and MitoTracker assays for ferroptosis-related metrics, we found that luteolin enhances human umbilical vein endothelial cells (HUVECs) antioxidant damage capacity. Drug affinity responsive target stability (DARTS), surface plasmon resonance (SPR), computer simulated docking and western blot showed that heat shock protein beta-1 (HSPB1) is one of the targets of luteolin action. Luteolin inhibits ferroptosis by promoting the protein expression of HSPB1/solute carrier family 7 member 11 (SLC7A11)/ glutathione peroxidase 4 (GPX4). In conclusion, we have preliminarily elucidated the antioxidant damage ferroptosis ability and the target of action of luteolin to provide a theoretical basis for the application of luteolin in radiation injury diseases.

Nicorandil Regulates Ferroptosis and Mitigates Septic Cardiomyopathy via TLR4/SLC7A11 Signaling Pathway

This study mainly explored the role of nicorandil in regulating ferroptosis and alleviating septic cardiomyopathy through toll-like receptor (TLR) 4/solute carrier family 7 member 11 (SLC7A11) signaling pathway. Twenty-four male SD rats were randomly divided into control, Nic (nicorandil), LPS (lipopolysaccharide), and LPS + Nic groups and given echocardiography. A detection kit was applied to measure the levels of lactic dehydrogenase (LDH), cardiac troponin I (cTnI), and creatine kinase-MB (CK-MB); HE staining and the levels of glutathione (GSH), malondialdehyde (MDA), total iron, and Fe2+ of myocardial tissues were detected. Moreover, the expression of TLR4 and SLC7A11 were measured by qRT-PCR and the proteins regulating ferroptosis (TLR4, SLC7A11, GPX4, ACSL4, DMT1, Fpn, and TfR1) were checked by western blot. Myocardial cells (H9C2) were induced with lipopolysaccharide (LPS) and transfected with si-TLR4 or SLC7A11-OE. Then, the viability, ferroptosis, and TLR4/SLC7A11 signaling pathway of cells were examined. Nicorandil could significantly increase left ventricular (LV) ejection fraction (LVEF) while reduce LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV). Also, it greatly reduced the levels of LDH, cTnI, and CK-MB; alleviated the pathological changes of myocardial injury; notably decreased MDA, total iron, and Fe2+ levels in myocardial tissues; and significantly increased GSH level. Besides, nicorandil obviously raised protein levels of GPX4, Fpn, and SLC7A11, and decreased protein levels of ACSL4, DMT1, TfR1, and TLR4. After knockdown of TLR4 or overexpression of SLC7A11, the inhibition effect of nicorandil on ferroptosis was strengthened in LPS-induced H9C2 cells. Therefore, nicorandil may regulate ferroptosis through TLR4/SLC7A11 signaling, thereby alleviating septic cardiomyopathy.

Serine and arginine rich splicing factor1‑regulated microtubule interacting and trafficking domain containing1 affects colorectal cancer progression and ferroptosis.

As the third most common type of cancer globally, colorectal cancer (CC) is a prevalent digestive malignancy, with the second highest mortality rate among all types of cancer. It has been reported that microtubule interacting and trafficking domain containing 1 (MITD1) serves a pivotal role in the initiation and progression of diverse types of tumors. Nevertheless, the underlying mechanism of MITD1 in CC has not been previously investigated. The ENCORI and GEPIA databases were used to investigate the expression levels of MITD1 in patients with CC. Immunohistochemistry was used to detect the expression of MITD1 in cancer tissues obtained from patients with CC, while its mRNA and protein expression levels in CC cell lines were determined by reverse transcription-quantitative PCR and western blot analysis, respectively. Subsequently, MITD1 was knocked down in CC cells using an interference plasmid and Cell Counting Kit 8, colony formation, as well as EdU assays were performed to assess cell proliferation. Concurrently, wound healing and Transwell assays were performed to evaluate the migration and invasion abilities of CC cells. Lipid reactive oxygen species (ROS) levels were determined by BODIPY 581/591 C11 staining. In addition, the levels of oxidative stress markers and those of total iron were measured using the corresponding kits. Furthermore, the association between serine and arginine rich splicing factor 1 (SRSF1) and MITD1 was verified by RNA immunoprecipitation and actinomycin D experiments. Finally, to further uncover the mechanism of MITD1, SRSF1 was overexpressed and MITD1 was silenced in CC cells. The results demonstrated that the expression of MITD1 was abnormally elevated in CC tissues and CC cell lines. MITD1 silencing distinctly diminished CC cell viability, increased CC cell ferroptosis and attenuated their invasion and migration abilities. In addition, MITD1 knockdown significantly increased the expression of lipid ROS and total iron levels in CC cells. Additionally, the results showed that SRSF1 could stabilize MITD1 mRNA expression in CC cells. Finally, it was revealed that SRSF1 could regulate MITD1 and affect the progression of CC and ferroptosis via p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling. Overall, the results of the current study indicated that SRSF1-regulated MITD1 could affect CC progression and ferroptosis, probably via the p53/SLC7A11/GPX4 signaling pathway.

Abstract P5-12-01: MUTYH In A Cancer Population

Abstract Background: Germline genetic testing has become a critical quality point in caring for breast cancer patients and high risk patients. Originally testing panels were extremely limited, but now expanded hereditary cancer testing panels are more common and have raised the question of other genes being linked to cancer risk. In this cohort, we examine the prevalence of the gene mutation MUTYH in a high risk population tested with expanded panels. Method: Patient data was obtained from an IRB-approved multi-center longitudinal, observational study, in which 2276 patients underwent expanded (>9 gene) germline genetic testing and also contributed personal and family history of cancer information. The average age in this cohort was 58.58 years old, with 2197 (96.53%) Female and 1762 (77.43%) with a personal cancer diagnosis and 1625 (71.40%) with a family history of cancer. Germline genetic tests were lab agnostic and tested an average of 73.2 genes (1214 or 53% tested with 85 gene panel) and a range of 9-85 genes tested. Results: Overall, the patients had a 16.70% positivity rate for pathogenic germline result of genetic test, with 42 (1.85%) reporting a Monoallelic MUTYH pathogenic variant (PV) result. Of those reporting a MUTYH PV, the average age was 58.48, with 40 (95.24%) females and 35 (83.33%) with a personal cancer diagnosis and 32 (76.19%) with a family history of cancer. Those patients with and without a personal and family history of cancer were compared, and found that a personal history of cancer has a very significant difference in MUTYH PV rate (9.56e-6) while family history of cancer does not have a significant difference (0.496). In the patients with a MUTYH PV and a diagnosis of cancer, 31 (88.57%) had a breast cancer diagnosis and only 1 (2.86%) had a colorectal cancer diagnosis - 73.81% and 2.38% of all MUTYH carriers. Of the 32 patients who had information about their family cancer diagnoses, there were 27 patients with multiple diagnoses and only 5 with a single family diagnosis, with 116 total family members with reported diagnosis. There were 44 breast cancer diagnoses in 23 of the MUTYH PV carriers’ families, which is 71.88% of all patients with family cancer information and 54.76% of all MUTYH PV carriers. There were 6 colorectal cancer diagnoses in 6 of the MUTYH PV carriers’ families, which is 18.75% of all patients with family cancer information and 14.29% of all MUTYH PV carriers. Conclusions: Our findings match with other reported cancer cohorts (on the order of 1-2%, Thompson et al). Monoallelic MUTYH has a significant association with both personal history of cancer. These findings suggest that patients with a personal and or family history of cancer should consider expanded gene panel testing which includes MUTYH. Requested Data 7/13 - MUTYH carrier personal and family history of cancer Citation Format: Linda Ann Smith. MUTYH In A Cancer Population [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-12-01.

Targeting fatty acid synthase modulates sensitivity of hepatocellular carcinoma to sorafenib via ferroptosis

BackgroundSorafenib resistance is a key impediment to successful treatment of patients with advanced hepatocellular carcinoma (HCC) and recent studies have reported reversal of drug resistance by targeting ferroptosis. The present study aimed to explore the association of fatty acid synthase (FASN) with sorafenib resistance via regulation of ferroptosis and provide a novel treatment strategy to overcome the sorafenib resistance of HCC patients.MethodsIntracellular levels of lipid peroxides, glutathione, malondialdehyde, and Fe2+ were measured as indicators of ferroptosis status. Biological information analyses, immunofluorescence assays, western blot assays, and co-immunoprecipitation analyses were conducted to elucidate the functions of FASN in HCC. Both in vitro and in vivo studies were conducted to examine the antitumor effects of the combination of orlistat and sorafenib and CalcuSyn software was used to calculate the combination index.ResultsSolute carrier family 7 member 11 (SLC7A11) was found to play an important role in mediating sorafenib resistance. The up-regulation of FASN antagonize of SLC7A11-mediated ferroptosis and thereby promoted sorafenib resistance. Mechanistically, FASN enhanced sorafenib-induced ferroptosis resistance by binding to hypoxia-inducible factor 1-alpha (HIF1α), promoting HIF1α nuclear translocation, inhibiting ubiquitination and proteasomal degradation of HIF1α, and subsequently enhancing transcription of SLC7A11. Orlistat, an inhibitor of FASN, with sorafenib had significant synergistic antitumor effects and reversed sorafenib resistance both in vitro and in vivo.ConclusionTargeting the FASN/HIF1α/SLC7A11 pathway resensitized HCC cells to sorafenib. The combination of orlistat and sorafenib had superior synergistic antitumor effects in sorafenib-resistant HCC cells.

BRCA1/2 Mutations in Vietnamese Patients with Hereditary Breast and Ovarian Cancer Syndrome.

(1) Background: Individuals with BRCA1/2 gene mutations are at increased risk of breast and ovarian cancer. The prevalence of BRCA1/2 mutations varies by race and ethnicity, and the prevalence and the risks associated with most BRCA1/2 mutations has not been unknown in the Vietnamese population. We herein screen the entire BRCA1 and BRCA2 genes for breast and ovarian cancer patients with a family history of breast cancer and ovarian cancer, thereby, suggesting a risk score associated with carrier status and history for aiding personalized treatment; (2) Methods: Between December 2017 and December 2019, Vietnamese patients who had a pathological diagnosis of breast and epithelial ovarian cancer were followed up, prospectively, after treatment from two large institutions in Vietnam. Blood samples from 33 Vietnamese patients with hereditary breast and ovarian cancers (HBOC) syndrome were collected and analyzed using Next Generation Sequencing; (3) Results: Eleven types of mutations in both BRCA1 (in nine patients) and BRCA2 (in three patients) were detected, two of which (BRCA1:p.Tyr1666Ter and BRCA2:p.Ser1341Ter) have not been previously documented in the literature. Seven out of 19 patient’s relatives had BRCA1/2 gene mutations. All selected patients were counselled about the likelihood of cancer rising and prophylactic screening and procedures. The study established a risk score associated with the cohorts based on carrier status and family history; (4) Conclusions: Our findings suggested the implications for the planning of a screening programme for BRCA1 and BRCA2 genes testing in breast and ovarian cancer patients and genetic screening in their relatives. BRCA1/2 mutation carriers without cancer should have early and regular cancer screening, and prophylactic measures. This study could be beneficial for a diverse group in a large population-specific cohort, related to HBOC Syndrome.

Interactions of Environmental Chemicals and Natural Products With ABC and SLC Transporters in the Digestive System of Aquatic Organisms.

An organism’s diet is a major route of exposure to both beneficial nutrients and toxic environmental chemicals and natural products. The uptake of dietary xenobiotics in the intestine is prevented by transporters of the Solute Carrier (SLC) and ATP Binding Cassette (ABC) family. Several environmental chemicals and natural toxins have been identified to induce expression of these defense transporters in fish and aquatic invertebrates, indicating that they are substrates and can be eliminated. However, certain environmental chemicals, termed Transporter-Interfering Chemicals or TICs, have recently been shown to bind to and inhibit fish and mammalian P-glycoprotein (ABCB1), thereby sensitizing cells to toxic chemical accumulation. If and to what extent other xenobiotic defense or nutrient uptake transporters can also be inhibited by dietary TICs is still unknown. To date, most chemical-transporter interaction studies in aquatic organisms have focused on ABC-type transporters, while molecular interactions of xenobiotics with SLC-type transporters are poorly understood. In this perspective, we summarize current advances in the identification, localization, and functional analysis of protective MXR transporters and nutrient uptake systems in the digestive system of fish and aquatic invertebrates. We collate the existing literature data on chemically induced transporter gene expression and summarize the molecular interactions of xenobiotics with these transport systems. Our review emphasizes the need for standardized assays in a broader panel of commercially important fish and seafood species to better evaluate the effects of TIC and other xenobiotic interactions with physiological substrates and MXR transporters across the aquatic ecosystem and predict possible transfer to humans through consumption.

Abstract 101: Studies on the flavonoid metabolite 2,4,6-trihydroxybenzoic acid as a CDK inhibitor and anti-proliferative agent: Potential role in cancer prevention

Abstract Colorectal cancer (CRC) is the second highest cause of cancer deaths in the United States according to Center for Disease Control. Increasing evidences suggest that a diet rich in fruits and vegetables will reduce incidences of colorectal adenomas. We hypothesize that the natural compounds present in fruits and vegetables, along with their metabolites generated by the gut microbiota, could be responsible for cancer prevention by targeting the cell cycle proteins. Previous studies conducted in the laboratory have identified 2,4,6- Trihydroxybenzoic acid (2,4,6-THBA), one of the degradation products of flavonoids, as a potential chemo-preventive agent mediating its effect through inhibition of cyclin dependent kinases (CDKs). Transport of monocarboxylic acids like 2,4,6-THBA is mediated by the Solute Ligand Carrier (SLC) family of transporters; in particular SLC5A8 and SLC5A12 have been implicated as tumor suppressor proteins that are down-regulated/non-functional in various cancers. In this study, we investigated the ability of the monocarboxylic acid transporter SLC5A8 to transport 2,4,6-THBA into the mammalian cells. Our pilot studies show that the cells expressing SLC5A8 transporters have the capacity to transport 2,4,6-THBA leading to upregulation of p21 and p27 as measured by western blots and qPCR. This was associated with decreased cell number as well as decreased colony formation as measured through clonogenic assays. We suggest that chemo-preventive actions of dietary compounds containing flavonoids may occur through 2,4,6-THBA, contributing largely to the prevention of CRC. Citation Format: Ranjini Sankaranarayanan, Chaitanya K. Valiveti, Severine van Slambrouck, Siddharth S. Kesharwani, D. Ramesh Kumar, Teresa Seefeldt, Hemachand Tummala, Jayarama B. Gunaje. Studies on the flavonoid metabolite 2,4,6-trihydroxybenzoic acid as a CDK inhibitor and anti-proliferative agent: Potential role in cancer prevention [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 101.

Sodium/glucose cotransporter 2 is expressed in choroid plexus epithelial cells and ependymal cells in human and mouse brains.

Diabetes mellitus (DM) is now recognized as one of the risk factors for Alzheimer's disease (AD), and the disease‐modifying effects of anti‐diabetic drugs on AD have recently been attracting great attention. Sodium/glucose cotransporter 2 (SGLT2) inhibitors are a new class of anti‐diabetic drugs targeting the SGLT2/solute carrier family 5 member 2 (SLC5A2) protein, which is known to localize exclusively in the brush border membrane of early proximal tubules in the kidney. However, recent data suggest that it is also expressed in other tissues. In the present study, we investigated the expression of SGLT2/SLC5A2 in human and mouse brains. Immunohistochemical staining of paraffin sections from autopsied human brains and C3H/He mouse brains revealed granular cytoplasmic immunoreactivity in choroid plexus epithelial cells and ependymal cells. Immunoblot analysis of the membrane fraction of mouse choroid plexus showed distinct immunoreactive bands at 70 and 26 kDa. Band patterns around 70 kDa in the membrane fraction of the choroid plexus were different from those in the kidney. Reverse transcription‐polymerase chain reaction analysis confirmed the expression of Slc5a2 mRNA in the mouse choroid plexus. Our results provide in vivo evidence that SGLT2/SLC5A2 is expressed in cells facing the cerebrospinal fluid, in addition to early proximal tubular epithelial cells. These findings suggest that SGLT2 inhibitors may have another site of action in the brain. The effects of SGLT2 inhibitors on brain function and AD progression merit further investigation to develop better treatment options for DM patients.

Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.

Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development.

Biallelic sequence and structural variants in RAX2 are a novel cause for autosomal recessive inherited retinal disease

PurposeRAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease.MethodsExome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members.ResultsBiallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry.ConclusionThis study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.

Impact of pathogenic mutations of the GLUT1 glucose transporter on channel dynamics using ConsDYN enhanced sampling

Background: The solute carrier (SLC) family of membrane proteins is a large class of transporters for many small molecules that are vital for the cell. Several pathogenic mutations are reported in the glucose transporter subfamily SLC2, causing Glut1-deficiency syndrome (GLUT1DS1, GLUT1DS2), epilepsy (EIG2) and cryohydrocytosis with neurological defects (Dystonia-9). Understanding the link between these mutations and transporter dynamics is crucial to elucidate their role in the dysfunction of the underlying transport mechanism. Methods: Predictions from SIFT and PolyPhen provided an impression of the impact upon mutation in the highly conserved RXGRR motifs, but no clear differentiation could be made by these methods between pathogenic and non-pathogenic mutations. Therefore, to identify the molecular effects on the transporter function, insight from molecular dynamic simulations is required. We studied a variety of pathogenic and non-pathogenic mutations, using a newly developed coarse-grained simulation approach ‘ConsDYN’, which allows the sampling of both inward-open and outward-occluded states. To guarantee the sampling of large conformational changes, we only include conserved restraints of the elastic network introduced upon coarse-graining, which showed similar reference distances between the two conformational states (≤1 Å difference). Results: We capture the ‘conserved dynamics’ between both states using ConsDYN. Simultaneously, it allowed us to considerably lower the computational costs of our study. This approach is sufficiently sensitive to capture the effect of different mutations, and our results clearly indicate that the pathogenic mutation in GLUT1, G91D, situated at the highly conserved RXGRR motif between helices 2 and 3, has a strong impact on channel function, as it blocks the protein from sampling both conformational states. Conclusions: Using our approach, we can explain the pathogenicity of the mutation G91D when we observe the configurations of the transmembrane helices, suggesting that their relative position is crucial for the correct functioning of the GLUT1 protein.

HAND2 loss-of-function mutation causes familial dilated cardiomyopathy

As two members of the basic helix-loop-helix family of transcription factors, HAND1 and HAND2 are both required for the embryonic cardiogenesis and postnatal ventricular structural remodeling. Recently a HAND1 mutation has been reported to cause dilated cardiomyopathy (DCM). However, the association of a HAND2 mutation with DCM is still to be ascertained. In this research, the coding regions and splicing junction sites of the HAND2 gene were sequenced in 206 unrelated patients affected with idiopathic DCM, and a new heterozygous HAND2 mutation, NM_021973.2: c.199G > T; p.(Glu67*), was discovered in an index patient with DCM. The nonsense mutation was absent in 300 unrelated, ethnically-matched healthy persons. Genetic scan of the mutation carrier's family members revealed that the genetic mutation co-segregated with DCM, which was transmitted in an autosomal dominant fashion, with complete penetrance. Functional deciphers unveiled that the mutant HAND2 protein had no transcriptional activity. In addition, the mutation abrogated the synergistic transcriptional activation between HAND2 and GATA4 or between HAND2 and NKX2.5, two other cardiac transcription factors that have been implicated in DCM. These research findings firstly suggest HAND2 as a novel gene predisposing to DCM in humans, which adds novel insight to the molecular pathogenesis of DCM, implying potential implications in the design of personized preventive and therapeutic strategies against DCM.

Functional monoamine oxidase B gene intron 13 polymorphism predicts putaminal dopamine turnover in de novo Parkinson's disease.

The objective of this study was to evaluate the effects of common functional polymorphisms in genes involved in dopamine metabolism on striatal dopamine turnover in de novo Parkinson's disease (PD). This was an observer-blinded cohort study investigating effects of common functional polymorphisms in dopa decarboxylase (DDC, rs921451), monoamine oxidase B (MAOB; rs1799836), catechol-O-methyltransferase (COMT, rs4680), and dopamine transporter/solute carrier family 6 member 3 (DAT/SLC6A3, variable number tandem repeats) genes on 18 F-fluorodopa uptake and an effective distribution volume ratio (inverse of dopamine turnover) measured by 18 F-fluorodopa PET in 28 untreated PD patients. Patients carrying the MAOBCC/(C)/CT genotype (low/intermediate enzyme activity) had a lower dopamine turnover in the putamen (higher mean effective distribution volume ratio) when compared with patients with MAOBTT/(T) genotype (high enzyme activity). Striatal PET measures were not different between variants in the remaining genes. The MAOB (rs1799836) polymorphism predicts putaminal dopamine turnover in early PD with the MAOBTT allele linked to high enzyme activity leading to higher intrinsic dopamine turnover, which has been demonstrated to constitute a risk factor for motor complications. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Pathway Analysis of a Transcriptome and Metabolite Profile to Elucidate a Compensatory Mechanism for Taurine Deficiency in the Heart of Taurine Transporter Knockout Mice

Taurine, which is abundant in mammalian tissues, especially in the heart, is essential for cellular osmoregulation. We previously reported that taurine deficiency leads to changes in the levels of several metabolites, suggesting that alterations in those metabolites might compensate in part for tissue taurine loss, a process that would be important in maintaining cardiac homeostasis. In this study, we investigated the molecular basis for changes in the metabolite profile of a taurine-deficient heart using pathway analysis based on the transcriptome and metabolome profile in the hearts of taurine transporter knockout mice (TauTKO mice), which have been reported by us. First, the genes associated with transport activity, such as the solute carrier (SLC) family, are increased in TauTKO mice, while the established transporters for metabolites that are elevated in the TauTKO heart, such as betaine and carnitine, are not altered by taurine deficiency. Second, the integrated analysis using transcriptome and metabolome data revealed significant increases and/or decreases in the genes involved in Arginine metabolism, Ketone body degradation, Glycerophospholipid metabolism, and Fatty acid metabolism in the KEGG pathway database. In conclusion, these pathway analyses revealed genetic compensatory mechanisms involved in the control of the metabolome profile of the taurine-deficient heart.

Study on p73 gene polymorphism and genetic susceptibility to hepatocellular cancer

Objective To evaluate the potential role of the separately p73 gene polymorphism on hepatocellular carcinoma(HCC) susceptibility. Methods A hospital-based case-control study was conducted in 100 cases HCC patients and 100 cases age, sex-matched cancer-free controls in the same region.The matrix-assisted laser desorption ionization time-of-flight mass spectrometry and DNA sequencing methods were used to analyze polymorphisms of p73 genetic polymorphisms (G4C14-A4T14). Using χ2 test to exam the differences of genotype between the HCC group and control group, logistic regression was used to analysis odds ratio(Oddsratios, OR) and 95%CI(Confidence Intervals, CI), adjusted for age and sex, analysis the association between SNPs of P73 polymorphisms and HCC.All the results were analyzed by SPSS 18.0. Results (1)The differences of age(χ2=0.185, P=0.667), gender(χ2=0.026, P=0.873), drinking status(χ2=0.427, P=0.514), and family history (χ2=0.058, P=0.809)of cancer composition between HCC group and control group were not statistically significant.(2)There were CC, CT and TT three genotypes in p73 rs1801173 C/T.The frequencies of CC, CT and TT genotypes of P73 C/T in controls and HCC cases were 65.0%, 28.2%, 6.8 % and 62.2%, 31.1%, 6.7%, respectively, and there was no significant difference between the two groups(χ2=0.326, P=0.568). (3)When stratifying by age, gender, smoking status, alcohol consumption, HBV carrier status and family history of cancer we found that the variant genotypes of GC/AT + AT/AT in P73 was associated with a significantly increased risk of HCC among HbsAg-positive individuals(χ2=3.916, P=0.048) and female(χ2=6.545, P=0.01), and there were no significant correlation with age, smoking, alcohol consumption, family history of cancer. Conclusion (1)p73 G4C14-to-A4T14 dinucleotide polymorphism may play a role in the development of chronic HBV-infected HCC in the Chinese population.(2)p73 G4C14-to-A4T14 dinucleotide polymorphism may play a role in the development of chronic HCC in female of Chinese population. Key words: Gene polymorphism; p73; Hepatocellular carcinoma