Osteosarcoma (OS) is the most common type of primary bone malignancy and has a poor prognosis. To investigate the mechanisms of osteosarcoma, the present analyzed the GSE28424 microarray. GSE28424 was downloaded from the Gene Expression Omnibus, and included a collective of 19 OS cell lines and four normal bone cell lines, which were used as controls. Subsequently, the differentially expressed genes (DEGs) were screened using the Limma package in Bioconductor. Gene Ontology (GO) and pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, interactions between the proteins encoded by the DEGs were identified using STRING, and the protein‑protein interaction (PPI) network was visualized using Cytoscape. In addition, modular analysis of the PPI network was performed using the Clique Percolation Method (CPM) in CFinder. A total of 1,170 DEGs were screened, including 530 upreguated and 640 downregulated genes. The enriched functions included organelle fission, immune response and response to wounding. In addition, RPL8 was observed to be involved with the ribosomal pathway in module A of the PPI network of the DEGs. PLCG1, SYK and PLCG2 were also involved in the B‑cell receptor signaling pathway in module B and the Fc‑epsilon RI signaling pathway in module C. In addition, AURKA (degree=39), MAD2L1 (degree=38), CDCA8 (degree=38), BUB1 (degree=37) and MELK (degree=37) exhibited higher degrees of connectivity in module F. The results of the present study suggested that the RPL8, PLCG1, PLCG2, SYK, MAD2L1, AURKA, CDCA8, BUB1 and MELK genes may be involved in OS.

Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca(2+) signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca(2+) signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca(2+) release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca(2+) influx through L-type Ca(2+) channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca(2+) release, resulting in increased VSM contraction in response to agonist stimulation.

Postmortem studies of people who have successfully committed suicide and people with depression have implicated the serotonin-1A (5-HT1A) receptor system in the pathophysiology of depression. Several molecular imaging studies have investigated alterations in 5-HT1A receptors in patients with depression using positron emission tomography and have reported conflicting results.

Allogeneic mesenchymal stem cell (MSC) transplantation improves cardiac function, but cellular differentiation results in loss of immunoprivilege and rejection. To explore the mechanism involved in this immune rejection, we investigated the influence of interleukin-6 (IL-6), a factor secreted by MSCs, on immune privilege after myogenic, endothelial and smooth muscle cell differentiation induced by 5-azacytidine, VEGF, and transforming growth factor-β (TGF-β), respectively. Both RT-PCR and ELISA showed that myogenic differentiation of MSCs was associated with significant downregulation of IL-6 expression (P < 0.01), which was also observed following endothelial (P < 0.01) and smooth muscle cell differentiation (P < 0.05), indicating that IL-6 downregulation was dependent on differentiation but not cell phenotype. Flow cytometry demonstrated that IL-6 downregulation as a result of myogenic differentiation was associated with increased leucocyte-mediated cell death in an allogeneic leucocyte co-culture study (P < 0.01). The allogeneic reactivity associated with IL-6 downregulation was also observed following MSC differentiation to endothelial and smooth muscle cells (P < 0.01), demonstrating that leucocyte-mediated cytotoxicity was also dependent on differentiation but not cell phenotype. Restoration of IL-6 partially rescued the differentiated cells from leucocyte-mediated cell death. These findings suggest that rejection of allogeneic MSCs after implantation may be because of a reduction in cellular IL-6 levels, and restoration of IL-6 may be a new target to retain MSC immunoprivilege.

Substantial evidence has shown that most exogenous substances are metabolized by multiple cytochrome P450 (P450) enzymes instead of by merely one P450 isoform. Thus, multi-P450 inhibition leads to greater drug-drug interaction risk than specific P450 inhibition. Herein, we innovatively established an artificial neural network cascade (NNC) model composed of 23 cascaded networks in a ladder-like framework to identify potential multi-P450 inhibitors among natural compounds by integrating 12 molecular descriptors into a P450 inhibition score (PIS). Experimental data reporting in vitro inhibition of five P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were obtained for 8,148 compounds from the Cytochrome P450 Inhibitors Database (CPID). The results indicate significant positive correlation between the PIS values and the number of inhibited P450 isoforms (Spearman's ρ = 0.684, p < 0.0001). Thus, a higher PIS indicates a greater possibility for a chemical to inhibit the enzyme activity of at least three P450 isoforms. Ten-fold cross-validation of the NNC model suggested an accuracy of 78.7% for identifying whether a compound is a multi-P450 inhibitor or not. Using our NNC model, 22.2% of the approximately 160,000 natural compounds in TCM Database@Taiwan were identified as potential multi-P450 inhibitors. Furthermore, chemical similarity calculations suggested that the prevailing parent structures of natural multi-P450 inhibitors were alkaloids. Our findings show that dissection of chemical structure contributes to confident identification of natural multi-P450 inhibitors and provides a feasible method for virtually evaluating multi-P450 inhibition risk for a known structure.

TRPMLs (or mucolipins) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the TRPML family includes three members: TRPML1-3. Although TRPML1 and TRPML3 have been well characterized, the cellular function of TRPML2 has remained elusive. To address TRPML2 function in a physiologically relevant cell type, we first analyzed TRPML2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of TRPML2 at the transcriptional level. Although TRPML2 expression was negligible in resting macrophages, TRPML2 mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, TRPML1 and TRPML3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous TRPML2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with TRPML1 and TRPML3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of TRPML2, we generated a TRPML2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in TRPML2-knockout mice. Furthermore, TRPML2-knockout mice displayed impaired recruitment of peripheral macrophages in response to i.p. injections of LPS or live bacteria, suggesting a potential defect in the immune response. Overall, our study reveals interesting differences in the regulation and distribution of the members of the TRPML family and identifies a novel role for TRPML2 in the innate immune response.

Growing evidences support the concept that peritumoral microenvironment gene expression is an important element for physicians to make an accurate prognosis. Nonetheless, the correlation between peritumoral ubiquitin ligases and the hepatocellular carcinoma (HCC) survival remains unclear till this present. The expression of intratumoral and peritumoral Casitas B-lineage Lymphoma (Cbl) and epidermal growth factor receptor (EGFR) in hepatocellular carcinomas (HCCs) followed by curative resection was assessed by tissue microarray-based immune-histochemistry in two independent cohorts (n = 352). Their respective prognostic values and other clinicopathologic factors were then evaluated. The peritumoral Cbl density, much higher than that in intratumoral tissue, was an independent prognostic factor for overall survival (P < 0.001) and time to recurrence (P < 0.001) of HCCs after curative resection. The hazard ratio were 1.587 and 1.689, respectively. However, there was no correlation between intratumoral Cbl and prognosis. The peritumoral Cbl was also associated with prognosis even in HCC subgroups with small tumor size, negative AFP, without microvascular invasion and negative HBeAg. After a thorough analysis pertaining to the key role of Cbl on ubiquitination and degradation of activated receptor tyrosine kinases, we eventually discovered the negative correlation between peritumoral Cbl and EGFR (P = 0.015). Furthermore, the combination of peritumoral Cbl and EGFR serves as a much stronger indicator to make an accurate prognosis, especially during early recurrence (P < 0.001). These findings suggest that low expression of peritumoral Cbl and EGFR were positively associated with tumor size, microvascular invasion and patients survival after hepatectomy, highlighting the key role of peritumoral liver milieu in HCC progression.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Significantly downregulated histidine-rich glycoprotein (HRG) during the dynamic stages (WB, WB7, and WB11) of neoplastic transformation of WB F344 hepatic oval-like cells was screened out by iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. HRG expression was significantly lower in HCC tissues. HRG overexpression in Huh7 and MHCC-97H hepatoma cell lines led to decreased cell proliferation, colony-forming ability, and tumor growth, and increased cell apoptosis. HRG could inhibit cell proliferation via the FGF-Erk1/2 signaling pathway by reducing Erk1/2 phosphorylation. On the other hand, the functional expression of HRG was also dependent on the glycosylation status at its N-terminal, especially at the glycosylation site Asn 125. The glycosylation of HRG may play a key competitive role in the interaction between HRG and heparin sulfate for binding bFGF and activating the FGF receptor. These findings provide novel insights into the molecular mechanism of HRG in HCC.

A series of linezolid analogues containing a hydrazone moiety were designed, synthesized and evaluated for their antibacterial activity. Most compounds exhibited more potent antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens as compared with linezolid and radezolid. Compounds 9a, 9c, 9f, 9g, 10m and 10t were more potent against tested clinical isolates of MRSA, MSSA, VRE and LREF as compared to linezolid. Compound 9a exhibited comparable activity with linezolid against human MAO-A for safety evaluation and showed moderate metabolism in human liver microsome. The most promising compound 9a showed remarkable antibacterial activity against S.aureus, MRSA, MSSA, LREF and VRE pathogens with MIC value of 0.0675 mg/mL, respectively, which was 15- to 30-fold more potent than linezolid.

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca2+-activated Cl- channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-β1/Smad3, and non-canonical TGF-β1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl--sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-β1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

microRNAs (miRNAs) play an important role in carcinogenesis. Typically, miRNAs downregulate the target expression by binding to the 3' UTR of mRNAs. However, recent studies have demonstrated that miRNAs can upregulate target gene expression, but its mechanism is not fully understood. We previously found that G-rich RNA sequence binding protein (GRSF1) mediates upregulation of miR-346 on hTERT gene. To explore whether GRSF1 mediate other miRNA's upregulation on their target genes, we obtained profile of GRSF1-bound miRNAs by Flag-GRSF1-RIP-deep sequencing and found 12 novel miRNAs, named miR-G. In this study, we focused on miR-G-10, which is highly expressed in cervical cancer tissues and cell lines and serum from patients with metastatic cervical cancer. miR-G-10 in cervical cancer cells significantly promoted migration/invasion and anoikis resistance in vitro and lung metastasis in vivo. Furthermore, miR-G-10 bound to the 3' UTR of PIK3R3 and upregulated its expression to activate the AKT/NF-κB signal pathway in a GRSF1-dependent manner, whereas miR-G-10 suppressed TIMP3 in the AGO2 complex to modulate the MMP9 signaling pathway in cervical cancer cells. Taken together, our findings may provide a new insight into the upregulation mechanism mediated by miRNAs and a potential biomarker for cervical cancer.

Glycyrrhizic acid (GA), the major bioactive triterpene glycoside of glycyrrhiza, has been shown to possess a wide range of pharmacological properties, including anti-inflammatory and anti-viral properties. However, few studies have examined the anti-allergic activity and exact mechanism of action of GA. In the present work, the anti-allergic activity and possible mechanisms of action of GA on an immunoglobulin (Ig) E-mediated allergic reaction has been studied using three models of allergic reaction in vivo and in vitro. Active systemic allergic reaction in Balb/c mice showed that GA can suppress the increased level of IL-4 to restore the immune balance of TH1/TH2 cells in a dose-dependent manner. Additionally, GA attenuated significantly the B cells producing allergen-specific IgE and IgG1 partly because of the low levels of TH2 cytokines. Both passive cutaneous anaphylaxis in vivo and an RBL-2H3 cell-based immunological assay in vitro indicated that GA acted as a "mast cell stabilizer", as it inhibited mast cell degranulation and decreased vascular permeability by inhibiting the expression of Orai1, STIM1 and TRPC1, which blocked extracellular Ca2+ influxes. The current study suggests that GA may serve as an effective anti-allergic agent derived from food for the prevention and treatment of IgE-mediated allergic reaction.

Recent animal studies have shown that mitochondrial dysfunction initiates and accelerates renal injury in sepsis, but its role in sepsis remains unknown. Mitochondrial stress or dying cells can lead to fragmentation of the mitochondrial genome, which is considered a surrogate marker of mitochondrial dysfunction. Therefore, we evaluated the efficiency of urinary mitochondrial DNA (UmtDNA) as a marker of renal dysfunction during sepsis-induced acute kidney injury (AKI).

Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-L-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-L-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. KEY POINTS: • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

In this study we have tested the efficacy of citrate therapy in various cancer models. We found that citrate administration inhibited A549 lung cancer growth and additional benefit accrued in combination with cisplatin. Interestingly, citrate regressed Ras-driven lung tumors. Further studies indicated that citrate induced tumor cell differentiation. Additionally, citrate treated tumor samples showed significantly higher infiltrating T-cells and increased blood levels of numerous cytokines. Moreover, we found that citrate inhibited IGF-1R phosphorylation. In vitro studies suggested that citrate treatment inhibited AKT phosphorylation, activated PTEN and increased expression of p-eIF2a. We also found that p-eIF2a was decreased when PTEN was depleted. These data suggest that citrate acts on the IGF-1R-AKT-PTEN-eIF2a pathway. Additionally, metabolic profiling suggested that both glycolysis and the tricarboxylic acid cycle were suppressed in a similar manner in vitro in tumor cells and in vivo but only in tumor tissue. We reproduced many of these observations in an inducible Her2/Neu-driven breast cancer model and in syngeneic pancreatic tumor (Pan02) xenografts. Our data suggests that citrate can inhibit tumor growth in diverse tumor types and via multiple mechanisms. Dietary supplementation with citrate may be beneficial as a cancer therapy.

In fluorosis-endemic areas, exposure to high levels of fluoride causes neurotoxicity such as lowered intelligence and cognitive impairment. Oxidative damage is critical to pathophysiologic processes of fluoride intoxication, and neurotoxicity of fluoride may be associated with oxidative stress. In previous studies, maize purple plant pigment (MPPP), which was rich in anthocyanins, showed a strong scavenging activity in vitro and in vivo. The present study aimed to determine whether treatment with MPPP can alleviate fluoride-induced oxidative damage in rat brain. After 3 months of experiment, brain tissues were assayed for oxidative stress variables, histological and Western blotting examinations. Our results showed that MPPP reduced the elevated malondialdehyde levels, increased superoxide dismutase activity, and further attenuated histopathological alterations and mitigated neuronal apoptosis. Importantly, MPPP also reversed changes in Bax and Bcl-2. Therefore, it was speculated that MPPP protects brain tissue from fluoride toxicity through its antioxidant capacity.

Long non-coding RNAs (lncRNAs) are essential contributors to the progression of various human cancers. Long intergenic non-protein coding RNA 1106 is a member of lncRNAs family. Until now, the specific role of LINC01106 in CRC remains undefined. The aim the current study was to unveil the functions of LINC01106 and explore its potential molecular mechanism in CRC. Based on the data of online database GEPIA, we determined that LINC01106 was expressed at a high level in colon adenocarcinoma (COAD) tissues compared to normal colon tissues. More importantly, high level of LINC01106 had negative correlation with the overall survival of COAD patients. Additionally, we also determined the low level of LINC01106 in normal colon tissues based on UCSC database. Through qRT-PCR, we identified that LINC01106 was highly expressed in CRC tissues compared to adjacent normal ones. Similarly, we detected the expression of LINC01106 and confirmed that LINC01106 was expressed higher in CRC cells than that in normal cells. Subsequently, LINC01106 was mainly distributed in the cytoplasm. LINC01106 induced the proliferation, migration, and stem-like phenotype of CRC cells. Mechanistically, cytoplasmic LINC01106 positively modulated Gli4 in CRC cells by serving as a miR-449b-5p sponge. Furthermore, nuclear LINC01106 could activate the transcription of Gli1 and Gli2 through recruiting FUS to Gli1 and Gli2 promoters. Mechanism of investigation unveiled that Gli2 was a transcription activator of LINC01106. In conclusion, Gli2-induced upregulation of LINC01106 aggravates CRC progression through upregulating Gli2, Gli2, and Gli4.

Radiotherapy with or without concurrent chemotherapy is the standard treatment for nasopharyngeal carcinoma (NPC) patients, whose efficacy is limited partly by intrinsic and acquired radioresistance. DNA methyltransferase 3B (DNMT3B) has been reported to participate in tumorigenesis via DNA methylation, but its role in mediating progression and radioresistance of NPC remains unclear. Therefore, we conducted the following studies to explore the relationship between DNMT3B and NPC. Here, we found that DNMT3B was elevated in NPC tissues and predicted the poor prognosis of NPC patients. We demonstrated for the first time that ionizing radiation could induce DNMT3B, which might be one of the reasons for radioresistance. Silencing of DNMT3B inhibited migration and invasion via suppressing epithelial-mesenchymal transition (EMT) in NPC cells. Furthermore, silencing DNMT3B restored and activated p53 and p21 via DNA demethylation, which led to cell cycle arrest and apoptosis, resulting in increased radiosensitivity of NPC both in vitro and in vivo. DNMT3B functions as a novel oncogene in the radioresistance of NPC through regulating EMT, cell cycle, and apoptosis. Therefore, DNMT3B could be a potential target for NPC treatment.

Drug-induced hepatitis (DIH), which seriously interferes with disease treatment, is one of the most common reasons for termination of new drugs during preclinical studies or post-marketing surveillance. Although antioxidants and anti-inflammatory agents are promising, their nonspecific distribution and insolubility limit their application. Therefore, precise drug release at the disease site is an important way to alleviate DIH and avoid side effects. Methods: A gripper-like hydrophilic cyclic phenylboronic acid (cPBA) was synthesized and a nanoprodrug (cPBA-BE) was established by coupling cPBA with hydrophobic baicalein (BE). The stimuli-responsive release properties and therapeutic effect of cPBA-BE on drug-injured hepatocyte were investigated. The biodistribution and therapeutic effect of cPBA-BE both in acetaminophen-induced acute hepatitis model and rifampicin-induced chronic hepatitis model were further evaluated. Results: cPBA-BE conjugate could self-assemble into nanoprodrug with cPBA as the hydrophilic external layer and BE as the hydrophobic core. In HepaRG cells, cPBA-BE showed stronger cellular uptake. Due to the H2O2- and acid-sensitivity, cPBA-BE could achieve adequate BE release, significantly resist the depletion of GSH, mitochondrial dysfunction, downregulation of inflammation and cell apoptosis in the acetaminophen injured HepaRG cells. Biodistribution showed that cPBA-BE specifically increased the concentration of BE in the liver of DIH mice. cPBA-BE could alleviate acetaminophen-induced acute hepatitis or rifampicin-induced chronic hepatitis more effectively through relieving the oxidative stress, inflammation and block the neutrophil infiltration in liver. Conclusions: cPBA is expected to be a good platform for constructing injectable nanoprodrug with both H2O2 and pH-responsive properties by coupling a wide range of drugs containing o-diol. In this study, the nanoprodrug cPBA-BE was determined to be effective for alleviating the DIH.

Store-operated Ca2+ entry (SOCE) represents a predominant Ca2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient's lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID.