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γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

Endoplasmic reticulum (ER) stress response is an adaptive program to cope with cellular stress that disturbs the function and homeostasis of ER, which commonly occurs during cancer progression to late stage. Late-stage cancers, mostly requiring chemotherapy, often develop treatment resistance. Chemoresistance has been linked to ER stress response; however, most of the evidence has come from studies that correlate the expression of stress markers with poor prognosis or demonstrate proapoptosis by the knockdown of stress-responsive genes. Since ER stress in cancers usually persists and is essentially not induced by genetic manipulations, we used low doses of ER stress inducers at levels that allowed cell adaptation to occur in order to investigate the effect of stress response on chemoresistance. We found that prolonged tolerable ER stress promotes mesenchymal-epithelial transition, slows cell-cycle progression, and delays the S-phase exit. Consequently, cisplatin-induced apoptosis was significantly decreased in stress-adapted cells, implying their acquisition of cisplatin resistance. Molecularly, we found that proliferating cell nuclear antigen (PCNA) ubiquitination and the expression of polymerase η, the main polymerase responsible for translesion synthesis across cisplatin-DNA damage, were up-regulated in ER stress-adaptive cells, and their enhanced cisplatin resistance was abrogated by the knockout of polymerase η. We also found that a fraction of p53 in stress-adapted cells was translocated to the nucleus, and that these cells exhibited a significant decline in the level of cisplatin-DNA damage. Consistently, we showed that the nuclear p53 coincided with strong positivity of glucose-related protein 78 (GRP78) on immunostaining of clinical biopsies, and the cisplatin-based chemotherapy was less effective for patients with high levels of ER stress. Taken together, this study uncovers that adaptation to ER stress enhances DNA repair and damage tolerance, with which stressed cells gain resistance to chemotherapeutics.

Background: Aberrant fucosylation plays a critical role in lung cancer progression. Nevertheless, the key fucosyltransferase with prognostic significance in lung cancer patients, the enzyme's intracellular targets, and complex molecular mechanisms underlying lung cancer metastasis remain incompletely understood., Methods: We performed a large-scale transcriptome-clinical correlation to identify major fucosyltransferases with significant prognostic values. Invasion, migration, cell adhesion assays were performed using lung cancer cells subject to genetic manipulation of FUT4 levels. Genome-wide RNA-seq and immunoprecipitation-mass spectrometry were used to characterize major cellular processes driven by FUT4, as well as profiling its intracellular protein targets. We also performed lung homing and metastasis assays in mouse xenograft models to determine in vivo phenotypes of high FUT4-expressing cancer cells., Findings: We show that FUT4 is associated with poor overall survival in lung adenocarcinoma patients. High FUT4 expression promotes lung cancer invasion, migration, epithelial-to-mesenchymal transition, and cell adhesion. FUT4-mediated aberrant fucosylation markedly activates multiple cellular processes, including membrane trafficking, cell cycle, and major oncogenic signaling pathways. The effects are independent of receptor tyrosine kinase mutations. Notably, genetic depletion of FUT4 or targeting FUT4-driven pathways diminishes lung colonization and distant metastases of lung cancer cells in mouse xenograft models., Interpretation: We propose that FUT4 can be a prognostic predictor and therapeutic target in lung cancer metastasis. Our data provide a scientific basis for a potential therapeutic strategy using targeted therapy in a subset of patients with high FUT4-expressing tumors with no targetable mutations., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The primary cilium plays an important role in mechanosensation in mammalian cells. To understand mechanosensation in the primary cilium, we combined a microfluidic device with super-resolution microscopy to study the primary cilium phenotypes. The microfluidic system enabled the precise control of the flow shear within a well-confined cell-culture environment. In addition, in situ cilia fixation was possible by switching from the culture medium to the fixation buffer instantaneously, which preserved the real-time cilium phenotype under the flow shear. After fixation, multiple cilium-specific proteins were immunostained to quantify the cilia bending behavior. We found that >50% of the primary cilia of mouse inner medullary collecting duct cells were highly aligned with the direction of flow under 11 Pa shear stress. Finally, we used super-resolution microscopy to observe the redistribution of two major cilium-specific proteins under flow shear, acetylated alpha-tubulin, and intraflagellar transport protein 88. To the best of our knowledge, this is the first platform to combine a microfluidic device with super-resolution microscopy to enable flow stimulation and in situ fixation for the observation of ciliary protein. This system can potentially be applied to the future development of a stimulation-enabled organ-on-a-chip to observe the intercellular signaling of primary cilia or for the analysis of disease mechanisms associated with ciliary mutations at the organ level.

The primary cilium is an essential organelle mediating key signaling activities, such as sonic hedgehog signaling. The molecular composition of the ciliary compartment is distinct from that of the cytosol, with the transition zone (TZ) gated the ciliary base. The TZ is a packed and organized protein complex containing multiple ciliopathy-associated protein species. Tectonic 2 (TCTN2) is one of the TZ proteins in the vicinity of the ciliary membrane, and its mutation is associated with Meckel syndrome. Despite its importance in ciliopathies, the role of TCTN2 in ciliary structure and molecules remains unclear. Here, we created a CRISPR/Cas9 TCTN2 knockout human retinal pigment epithelial cell line and conducted quantitative analysis of geometric localization using both wide-field and super-resolution microscopy techniques. We found that TCTN2 depletion resulted in partial TZ damage, loss of ciliary membrane proteins, leakage of intraflagellar transport protein IFT88 toward the basal body lumen, and cilium shortening and curving. The basal body lumen occupancy of IFT88 was also observed in si-RPGRIP1L cells and cytochalasin-D-treated wild-type cells, suggesting varying lumen accessibility for intraflagellar transport proteins under different perturbed conditions. Our findings support two possible models for the lumen leakage of IFT88, i.e., a tip leakage model and a misregulation model. Together, our quantitative image analysis augmented by super-resolution microscopy facilitates the observation of structural destruction and molecular redistribution in TCTN2 -/- cilia, shedding light on mechanistic understanding of TZ-protein-associated ciliopathies., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.

Monoclonal antibodies enhance innate immunity, while bispecific T cell engager antibodies redirect adaptive T cell immunity. To stimulate both innate and adaptive mechanisms, we created a bifunctional eCD16A/anti-CD3-BFP adapter protein for combined use with clinically approved monoclonal IgG1 antibodies. The adaptor protein contains the extracellular domain of the human CD16A high-affinity variant, which binds the Fc domain of IgG1 antibodies, and an anti-human CD3 single-chain variable fragment that redirects T cell cytotoxicity. Functional characterization of eCD16A/anti-CD3-BFP was performed using in vitro , ex vivo , and in vivo animal assays. Combination treatments of eCD16A/anti-CD3-BFP with rituximab (anti-CD20), cetuximab (anti-EGFR), trastuzumab (anti-HER2) or anti-PD-L1 IgG1 led to specific killing of antigen-expressing tumor cells. In an ex vivo assay, eCD16A/anti-CD3-BFP combined with rituximab effectively eliminated B cells within peripheral blood samples from healthy donors and cancer patients; the effectiveness of this treatment was further enhanced by addition of either autologous or allogeneic γ9δ2 T cells. In mouse xenograft studies, the combination of eCD16A/anti-CD3-BFP and rituximab led to rapid depletion of tumor cells. The presence of non-targeting competing serum immunoglobulins interfered with efficacy, but this interference could be overcome by eCD16A/anti-CD3-BFP dose escalation or by using a Fc-glycoengineered version of rituximab. Moreover, combining eCD16A/anti-CD3-BFP with anti-Epstein Barr virus (EBV) antibodies directed T cell cytotoxicity toward EBV-infected cells. These findings support further development of eCD16A/anti-CD3-BFP as a novel anti-cancer therapeutic to stimulate both innate and adaptive immunity.

Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

Halobacterium sp. NRC-1 is a wild-type extremophilic microbe that is naturally tolerant to high levels of ionizing radiation. Mutants of strain NRC-1 with even higher levels of resistance to ionizing radiation, named RAD, were previously isolated after selecting survival to extremely high doses of ionizing radiation. These RAD mutants displayed higher transcription levels for the rfa3 operon, coding two subunits of the RPA-like putative single-stranded binding protein, rfa3 and rfa8, and a third downstream gene, ral. In order to bioengineer cells with increased tolerance to ionizing radiation and further explore the genetic basis of the RAD phenotype, we placed the rfa3 operon under control of the gvpA promoter in a Halobacterium expression plasmid, pDRK1. When pDRK1 was introduced into the wild-type NRC-1 strain, overproduction of the Rfa3 and Rfa8 proteins was observed by Western blotting and proteomic analysis. The Halobacterium strains expressing Rfa3 and Rfa8 also displayed improved survival after exposure to ionizing radiation, similar to the RAD mutants, when compared to wild-type strain NRC-1. The Rfa3 and Rfa8 proteins co-purified by affinity chromatography on single-stranded DNA cellulose columns, confirming the ability of the proteins to bind to single-stranded DNA as well as their relative abundance in the wild-type, RAD mutants, and rfa3 operon overexpression strains. These results clearly establish that overexpression of haloarchaeal RPA promotes ionizing radiation resistance in Halobacterium sp. NRC-1 and that the Rfa3 and Rfa8 subunits bind single-stranded DNA. Bioengineering cells with increased levels of ionizing radiation resistance may have potential value in medical and environmental applications.

Halobacterium salinarum is an extremely halophilic archaeon that inhabits high-salinity aqueous environments in which the temperature can range widely, both daily and seasonally. An OMICS analysis of the 37°C and 49°C proteomes and transcriptomes for revealing the biomodules affected by temperature is reported here. Analysis of those genes/proteins displaying dramatic changes provided a clue to the coordinated changes in the expression of genes within five arCOG biological clusters. When proteins that exhibited minor changes in their spectral counts and insignificant p values were also examined, the apparent influence of the elevated temperatures on conserved chaperones, metabolism, translation, and other biomodules became more obvious. For instance, increases in all eight conserved chaperones and three arginine deiminase pathway enzymes and reductions in most tricarboxylic acid (TCA) cycle enzymes and ribosomal proteins suggest that complex system responses occurred as the temperature changed. When the requirement for the four proteins that showed the greatest induction at 49°C was analyzed, only CctA (chaperonin subunit α), but not Hsp5, DpsA, or VNG1187G, was essential for thermotolerance. Environmental stimuli and other perturbations may induce many minor gene expression changes. Simultaneous analysis of the genes exhibiting dramatic or minor changes in expression may facilitate the detection of systems level responses.

Mass measurement and precursor mass assignment are independent processes in proteomic data acquisition. Due to misassignments to C-13 peak, or for other reasons, extensive precursor mass shifts (i.e., deviations of the measured from calculated precursor neutral masses) in LC-MS/MS data obtained with the high-accuracy LTQ-Orbitrap mass spectrometers have been reported in previous studies. Although computational methods for post-acquisition reassignment to monoisotopic mass have been developed to curate the MS/MS spectra prior to database search, a simpler method for estimating the fraction of spectra with precursor mass shift so as to determine whether the data require curation remains desirable. Here, we provide the evidence that an easy approach, which applies a large precursor tolerance (2.1Da or higher) in SEQUEST search against a forward and decoy protein sequence database and then filters the data with PeptideProphet peptide identification probability (p≥0.9), could detect most of the MS/MS spectra containing inaccurate precursor masses. Furthermore, through the implementation of artificial mass shifts on 4000 randomly selected MS/MS spectra, which originally had accurate precursor mass assigned by the mass spectrometers, we demonstrated that the accuracy of the precursor mass has almost negligible influence on the efficacy and fidelity of peptide identification., Biological Significance: Integral precursor mass shift is a known problem and thus proteomic data should be handled and analyzed properly to avoid losing important protein identification and/or quantification information. A quick and easy approach for estimating the number of MS/MS spectra with inaccurate precursor mass assignments would be helpful for evaluating the performance of the instrument, determining whether the data requires curation prior to database search or should be searched with specific search parameter(s). Here we demonstrated most of the MS/MS spectra with inaccurate mass assignments (integral or non-integral changes) that could be easily identified by database search with large precursor tolerance windows., (© 2013. Published by Elsevier B.V. All rights reserved.)

The use of induction therapy significantly reduces the incidence of acute rejection (AR) episodes posttransplantation and prevents delayed graft function (DGF). In our program, all adult deceased donor kidney transplant (DDKT) recipients receive immunosuppression induction therapy with either basiliximab (anti-CD25 Ab) or rabbit anti-thymocyte globulin (RATG). Our protocol is risk adjusted such that patients who are at a higher risk for DGF or AR received RATG and all other patients receive anti-CD25 Ab. We hypothesized that treating our higher-risk patients with RATG induction at the time of transplantation would lead to a lower rate of DGF and better outcomes. From August 1, 2005 through August 31, 2010, 116 consecutive adult patients received a DDKT in a single academic transplantation center. All DDKT patients received induction with RATG or anti-CD25 Ab. The induction decision was made prior to transplantation based on donor and recipient risk factors for AR and DGF. Transplants that were deemed at higher risk for DGF or AR based on donor factors or recipient factors received RATG. Medical records and patient databases were reviewed retrospectively. The use of RATG in higher-risk recipients for DGF and AR did not significantly reduce the DGF rate. At 6 months the function of the allograft function measured as creatinine clearance or serum creatinine was lower in the RATG group than the patients who received anti-CD25 Ab induction. The choice of induction therapy did not improve outcomes in high-risk patients in this short-term study., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The 1310 Haloarcula marismortui proteins identified from mid-log and late-log phase soluble and membrane proteomes were analyzed in metabolic and cellular process networks to predict the available systems and systems fluctuations upon environmental stresses. When the connected metabolic reactions with identified proteins were examined, the availability of a number of metabolic pathways and a highly connected amino acid metabolic network were revealed. Quantitative spectral count analyses suggested 300 or more proteins might have expression changes in late-log phase. Among these, integrative network analyses indicated approximately 106 were metabolic proteins that might have growth-phase dependent changes. Interestingly, a large proportion of proteins in affected biomodules had the same trend of changes in spectral counts. Disregard the magnitude of changes, we had successfully predicted and validated the expression changes of nine genes including the rimK, gltCP, rrnAC0132, and argC in lysine biosynthesis pathway which were downregulated in late-log phase. This study had not only revealed the expressed proteins but also the availability of biological systems in two growth phases, systems level changes in response to the stresses in late-log phase, cellular locations of identified proteins, and the likely regulated genes to facilitate further analyses in the postgenomic era.

Purpose: A case study of mycophenolate mofetil-induced oral ulcers in a renal transplant patient is reported., Summary: A 23-year-old Hispanic man who received a renal transplant from a living relative secondary to end-stage renal disease due to focal segmental glomerulosclerosis arrived at an outpatient clinic with gum swelling and pain. He had been on a maintenance immunosuppressive regimen consisting of cyclosporine 150 mg twice daily, mycophenolate mofetil 1 g twice daily, and prednisone 12.5 mg daily for approximately four months. Routine laboratory tests revealed an elevated serum creatinine concentration (2.2 mg/ dL) and a decreased white blood cell count (2.3 × 10(3)/μL). All other laboratory test values were within normal limits. Initially, cyclosporine-induced gingival hyperplasia was suspected. However, despite reduction of the cyclosporine dosage, the gum pain and swelling did not improve, and the patient began to complain of odynophagia and worsening of symptoms. On physical examination, scattered ulcerations on the gums and lips were noted. The diagnosis of oral ulcerations secondary to mycophenolate mofetil therapy was suspected when other etiologies, such as hematologic disorders, malignancies, and viral infections, were eliminated. Mycophenolate mofetil was discontinued. One week later, the patient's ulcers had regressed and odynophagia improved, as did his renal function and leukopenia. Mycophenolate mofetil was not restarted, and the patient reported complete resolution of symptoms six weeks after discontinuation of mycophenolate mofetil., Conclusion: After five months of therapy, a 23-year-old renal transplant recipient developed mycophenolate mofetil toxicity manifested as oral ulcers. Discontinuation of therapy resulted in rapid resolution of oral ulcers.

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.

Background: In an effort to improve our transplant program's dead-donor kidney acceptance criteria, we compared 2 different consecutive time periods in our transplant program. Period I, in which the program used more-restrictive criteria in accepting dead-donor kidneys for our patients, and period II, when the program used less-restrictive criteria for the dead-donor kidneys that were accepted. The less-restrictive criteria resulted in an increase in the number of renal transplants performed., Methods: A retrospective database analysis was performed of all organ-donor offers to a single kidney transplant program from July 1, 2004, to September 30, 2006 (period I = July 1, 2004, through July 10, 2005, and period II = July 11, 2005 through September 30, 2006). Kidney acceptance rates were compared between 2 consecutive time periods during which the program used different organ acceptance criteria. Data analysis included a comparison of donor characteristics, reason for organ refusal, creatinine clearance, and graft survival. Graft survival was obtained for both kidneys associated with each offer, even if 1 or both of the organs were transplanted at a different center., Results: Donor age and kidney quality were the most common reasons for refusal during both transplant periods. The organ acceptance rate improved markedly during period II. There was a marked increase in the number of kidney transplants performed during a 12-month period when comparing the 2 periods: 16 transplants during period I versus 46 transplants during period II. Graft survival was not significantly different between the 2 periods. Calculated creatinine clearance, which we used as a marker of organ quality, was statistically lower during period II., Conclusions: Increased acceptance rate was not associated with statistically significant decreased graft survival. Although an increase in delayed graft function was associated with broader acceptance criteria, this factor did not affect overall graft survival. By increasing our kidney acceptance rate, we were able to successfully transplant more patients.

A method for the separation and determination of terpineol oil by temperature programming capillary gas chromatography has been established. An OV-1 fused silica capillary column (30 m x 0.32 mm i.d. x 0.25 microns) was used with a temperature increase rates of 1.5 degrees C/min from 70 degrees C to 100 degrees C, 5 degrees C/min from 100 degrees C to 160 degrees C, 10 degrees C/min from 160 degrees C to 220 degrees C and then hold for 2 min. The main chemical components and their relative contents of the terpineol oil, terpinene oil, red and yellow oils of middle oils in the terpineol production and natural terpineol were analyzed by capillary gas chromatography-Fourier transform infrared spectrometry (GC-FTIR) and gas chromatography-mass spectrometry (GC-MS). The typical chromatograms of these oils are given. It has offered the information of the boiling points of the chemical compounds, and it is helpful to reutilize them after their separation by rectification. The results showed that the main compounds of the heavy cut of terpineol were longifolene and beta-caryophyllene. They are from the raw material, terpinene oil. The main compounds in the natural terpineol oil were eucalyptol, trans-4-thujanol, p-isopropenyl toluene, cis-4-thujanol, linalool, camphor, borneol, 4-terpineol, alpha-terpineol and safrole. The contents of beta-terpineol and gamma-terpineol were not so high as in synthetic terpineol.