Brain neoplasms constitute a group of tumors with discrete differentiation grades, and therefore, course of disease and prognosis. Magnetic resonance imaging (MRI) remains the gold standard method for the investigation of central nervous system tumors. However, MRI suffers certain limitations, especially if radiation therapy or chemotherapy has been previously applied. On the other hand, given the development of newer radiopharmaceuticals, positron emission tomography (PET) aims to a better investigation of brain tumors, assisting in the clinical management of the patients. In the present review, the potential contribution of radiolabeled fluorothymidine (FLT) imaging for the evaluation of brain tumors will be discussed. In particular, we will present the role of FLT-PET imaging in the depiction of well and poorly differentiated lesions, the assessment of patient prognosis and treatment response, and the recognition of disease recurrence. Moreover, related semi-quantitative and kinetic parameters will be discussed. Brain neoplasms can be classified into two main groups, primary tumors and metastatic brain lesions which are more common. Gliomas represent the most frequent type of primary brain tumors and mainly consist of malignant neoplasms; more than half of these lesions are glioblastomas . The inc18F-fluorodeoxyglucose (18F-FDG)—positron emission tomography (PET), the conventional PET imaging technique in oncology, has been also used in patients with brain lesions, either at their presentation, or for the assessment of response to treatment and detection of recurrence [18F-FDG is not considered as the most appropriate radiotracer for the investigation of brain lesions due to the high background activity. For this reason, several radiopharmaceuticals have been developed in this field, such as radiolabeled amino acids, 18F-choline, hypoxia detection agents and tumor proliferation markers (Table Magnetic resonance imaging (MRI) with gadolinium (Gd)—enhancement is the method of choice for the initial diagnostic investigation of brain lesions, as well as for the evaluation of treatment response and the early depiction of disease recurrence . 18F-flucurrence , 12. Not18F-FLT is a radiolabeled analog described initially as a selective inhibitor of DNA synthesis [18F-FDG enters cells by active transport through nucleoside transporters , as well as by passive diffusion [ynthesis . It was ynthesis , 15. Givynthesis . 18F-FDGiffusion . Howeveriffusion –18.18F-FLT uptake depends mainly on the increased permeability, intracellular transport and influx after the disruption of the blood–brain barrier (BBB) or the function of nucleoside transporters in case of intact BBB, whereas the contribution of the metabolic trapping through phosphorylation seems to be less important [18F-FLT uptake is the transport mechanism, while its accumulation is mainly attributed to the transport and influx rate [18F-FLT uptake and the metabolic rate (as described by K3 constant), indicating—at least in part—the contribution of the metabolic factor in radiotracers’ intracellular maintenance in this subgroup of tumors [18F-FLT could actually demonstrate cell proliferation in brain tumors. There is also a possibility to reflect only BBB disruption, a characteristic that could lead to controversy regarding radiotracer specificity in tumor detection. In particular, the non-specific binding of the radiotracer may be related to the false positive results, and could impair proper tumor delineation and characterization.In brain tumors, newer evidence suggests that mportant , 20. Thelux rate , 22. Intf tumors . Consequ18F-FLT (B) techniques.Accurate tumor detection and delineation, as well as grading before surgical resection, are of great importance. These parameters have influences on the surgical procedure, the post-surgical treatment management and the patient prognosis. In addition, it is crucial to identify tumor grade in inoperable cases. Figure 18F-FLT imaging has been reported to depict accurately the biopsy site when glioma is suspected [18F-FLT imaging is considered less valuable for low-grade gliomas, as they present no or little radiotracer uptake [18F-FLT imaging can provide important evidence in the discrimination of tumors between grades II and IV and grades III and IV, despite its lower performance for grade III vs. grade IV differentiation [uspected . The senuspected , 25, 26.uspected . In highr uptake . Neverthntiation .11C-methionine (11C-MET) imaging, the use of 18F-FLT leads to larger volume when delineating the tumor, compared to Gd-enhanced MRI method. Possibly, radiotracer accumulation may precede substantial BBB breakdown, or the radiotracer could be a more sensitive marker of BBB breakdown. Nevertheless, previous reports suggested a fairly good agreement in tumor volume definition between MRI and PET studies. The two diagnostic methods contribute supplementarily to the delineation of the tumor burden, even though tumor margins may be defined less accurately based on FLT uptake [In accordance with T uptake , 30, 31.18F-FLT, as expected based on the reported strong association between 18F-FLT uptake and Gd- enhancement [18F-FLT uptake or modifications in cerebral blood volume (CBV) maps. On the other hand, grade III gliomas usually demonstrate mild Gd-enhancement and radiotracer uptake, whereas grade IV gliomas show high Gd-enhancement and 18F-FLT uptake. However, a grade III glioma may demonstrate MRI and magnetic resonance spectroscopy (MRS) characteristics of glioblastoma multiform (including apparent diffusion coefficient (ADC)), despite the presence of mild 18F-FLT uptake, or a grade IV lesion may present non-profound perfusion changes though high radiotracer uptake [18F-FLT uptake has been related to tumor development, growth and size [Primary or progressive tumors with no or little enhancement in MRI images were found not to concentrate ancement , 32. Typr uptake . Moreover uptake . Finallyand size , 35.18F-FLT concentration remains constantly low in normal brain tissue allowing high tumor-to-background ratio, although SUV values may be low [18F-FLT), as well as in methods used to discriminate vascular and tissue activity. For high-grade lesions, no substantial differences have been observed between blood measurements from arterial blood sampling and PET derived venous measurements, indicating that such calculations can be easily applied in the clinical setting [18F-FLT PET findings in previously untreated low-grade gliomas were correlated with overall survival, not event-free survival, possibly due to referral bias [Notably, the standardized uptake value (SUV) calculations alone may not be adequate to assess the actual proliferative cellular activity. Uptake results could be also associated with non-specific leakage, probably representing BBB breakdown in high-grade gliomas. Therefore, the evaluation of compartmental-derived kinetic parameters has a significant role in this field . 18F-FLTy be low . Chen ety be low . Apparen setting . Regardi setting . In gene setting –38. Howeral bias .18F-FLT was proposed to be correlated with proliferation markers. After analyzing the results of previous studies with a total sample of 509 patients, Chalkidou et al. reported that 18F-FLT kinetic parameters and SUVmax values were associated with Ki-67 measurements [11C-MET, the association of 18F-FLT uptake with Ki-67 was found to be more significant, whereas the highest Ki-67 percentage glioblastoma cases exhibited high 18F-FLT, but moderate 11C-MET, uptake [18F-FLT uptake was significantly associated with Ki-67 both in newly diagnosed and recurrent brain tumors; however, the correlation in recurrent lesions was weaker [18F-FLT uptake and Ki-67 staining in both bevacizumab-treated and non-treated groups, suggesting an association between radiotracer uptake and angiogenesis inhibition [18F-FLT uptake were observed after irradiation of glioblastoma cells in a mouse model [18F-FLT PET imaging may provide additional information regarding tumor cell proliferation in radiation-treated areas. Finally, normal-to-background ratio in 18F-FLT imaging of newly diagnosed and recurrent tumors was positively correlated to the expression of a 58-kD microspherule protein highly produced in grade IV gliomas [Given its association with TK-1 at the salvage DNA synthesis pathway, urements . Moreoveurements . In comp, uptake . Furthers weaker . In a rahibition . Moreovese model . Consequ gliomas . Further gliomas .Pseudo-progression and pseudo-regression may present about two months after radiation therapy and temozolomide administration in patients with gliomas. Pseudo-progression refers to increased Gd-enhancement in MRI images despite response to treatment, whereas pseudo-regression corresponds to cases characterized by tumor progression despite decreased Gd-enhancement. Therefore, in the clinical setting, the main question is whether patient symptoms could be attributed to either recurrent disease or radiation necrosis .18F-FLT parameters in disease recurrence and treatment response. Since 18F-FLT uptake is mostly attributed to BBB disruption, imaging findings can be associated with necrosis after radiation therapy, or the presence of proliferating tissue [Studies not only in cell lines and animal models but also in humans have been performed investigating the role of semi-quantitative and dynamic kinetic g tissue . Further18F-FDG imaging, 18F-FLT was found to have higher sensitivity and accuracy in the investigation of disease recurrence, despite similar specificity [18F-FLT technique has lower ability to distinguish recurrent lesions according to their grade, than newly diagnosed tumors [max as a quantitative parameter in the differential diagnosis between radiation necrosis and disease recurrence, 18F-FLT technique yielded high sensitivity but moderate specificity, limiting its use as only a supplementary tool in this field. Although treatment-induced changes could be differentiated from recurrent lesions based on radiotracer uptake, as well as high- vs. low-grade recurrent lesions, the accumulation of 18F-FLT in low-grade gliomas is low  [18F-FLT imaging may be useful in radiation treatment planning, both for dose escalation in residual cancer cells and the protection of the surrounding normal tissue.Nevertheless, there is evidence that no currence . Notablynce [max . Further volume) . Consequ18F-FLT kinetic parameters were reported to perform better than semi-quantitative measurements in the differentiation between radiation necrosis and recurrent disease [max in tumor 18F-FLT kinetics assessment, compared to necrotic tissue characteristics due to radiation [max parameter [Finally,  disease . Particuadiation . On the arameter .18F-FLT technique, several uptake and kinetic parameters  have been investigated for the evaluation of treatment response and their ability to provide prognostic information. In a mouse model, the influence of radiation on the pattern and degree of radiotracer uptake was studied, showing important associations with micro-environmental changes in glioblastoma tumors [18F-FLT PET imaging may contribute to radiation therapy response assessment; however, clinical trials in this area are lacking [Depending on the location and differentiation state of each lesion, the therapeutic management of gliomas may include surgery, radiation therapy and/or chemotherapy. Subsequently, the evaluation of treatment response is of great importance since it is directly related to survival. Contrast enhancement MRI, with additional T2 and FLAIR-weighted MRI response assessment after chemotherapy, is the method of choice to evaluate treatment response, whereas MRS and radiolabeled amino acids PET imaging may be also helpful . Regardia tumors . Irradia lacking .18F-FLT technique has been demonstrated to provide useful information for the evaluation of response to chemotherapy, yielding prognostic value in newly diagnosed high-grade gliomas and recurrent brain tumors. After enrolling 21 patients with recurrent tumors treated with irinotecan-bevacizumab, Chen et al. demonstrated the predictive capability of the technique in depicting responders vs. non-responders and its correlation with progression-free and overall survival [survival . These fsurvival , 54. Intmax measurements. However, these discrepancies may be related to sample differences among studies. On the other hand, tumor-to-background measurements seemed to be a more reliable prognostic indicator [18F-FLT-derived proliferative volume was significantly correlated to overall survival [In everyday clinical practice, SUV values may be the only semi-quantitative measurements recorded . Notablyndicator , 56. Morsurvival . In compsurvival . Further18F-FLT kinetic parameters in patients with gliomas can be attributed to either tumor-induced consequences or changes related to treatment response; therefore, kinetic analysis may provide important prognostic information [18F-FLT kinetics may be useful in discriminating long-term vs. short-term survivors with high diagnostic accuracy, possibly leading to a more individualized therapeutic management [18F-FLT kinetic analysis was reported to predict overall survival more accurately in comparison to 18F-fluorodopa kinetics [18F-FLT kinetic data are obtained early in the treatment of recurrent brain tumors with bevacizumab and irinotecan, useful prognostic information can be obtained with reasonable confidence [Differences in ormation , 57. Accnagement . Moreovekinetics . Consequnfidence .MRI can offer morphological evidence in glioma patients. However, its value is limited for the evaluation of more specific information regarding the biological characteristics of the lesions. Moreover, MRI has certain limitations, particularly for the initial evaluation of tumor aggressiveness. False results have been also described, especially after radiotherapy or chemotherapy. Although advanced MRI techniques Fig.  may cont18F-FDG PET was initially proposed due to the increased glucose metabolism in high-grade gliomas, as well as the positive association between glycolysis rate and malignancy [18F-FDG uptake was linked to tumor grading, showing prognostic value [18F-FDG imaging is hampered by the high glucose metabolism in normal brain areas; both the sensitivity for tumor detection and specificity for tumor delineation are significantly limited [18F-FDG accumulation in inflammatory tissue makes the distinction between malignancy and inflammation often challenging. Regarding stereotactic biopsy target selection, 18F-FDG imaging was reported to be superior compared to MRI, despite its limited value in low-grade gliomas [lignancy . 18F-FDGic value . However limited . Particu limited . Moreove gliomas .18F-fluorocholine was considered to discriminate high-grade gliomas, metastatic lesions, and benign tumors. A main disadvantage is the high radiotracer accumulation in the choroid plexus, venous sinuses, and pituitary gland, limiting the value of the technique in the vicinity of these structures [Since cell proliferation can be related to higher metabolism of cell membrane components, radiolabeled choline was proposed for the assessment of brain lesions, particularly oligodendroglial tumors . Moreoveructures .18F-fluoromisonidazole, a marker of hypoxia, was observed in high grade but not in low-grade gliomas [Rapid tumor growth is associated with lower oxygen levels in parts of the lesion compared to the surrounding normal tissue, while hypoxia is linked to tumor progression and resistance to radiotherapy. Uptake of nitroimidazole derivative  gliomas . However18F-FET PET and MRI [Increased cell proliferation in gliomas leads to higher amino acid utilization . High-co and MRI . Further and MRI , 65. Fin and MRI .18F-FLT imaging focuses on the increased DNA replication observed in malignant transformation. Radiotracer uptake is lower in most regions because of the limited neuronal cell division [18F-FLT PET imaging can depict high-grade gliomas and assist in the discrimination between high- vs. low-grade lesions. Although this technique has been reported to depict the biopsy site, it cannot accurately identify tumor margins [18F-FLT imaging may assist in the investigation of recurrence after surgical excision (Fig. 18F-FLT uptake during treatment can provide valuable prognostic evidence, as well as information about treatment response.division . 18F-FLT margins , 31. On ion Fig. . Finally18F-FLT study. Notably, since cellular 18F-FLT uptake is limited by transport across the BBB, a complete kinetic model of radiotracer uptake, transport and metabolism could significantly improve DNA synthesis quantification. Furthermore, kinetic modeling may also provide valuable evidence for the discrimination between recurrence and radiation necrosis.Kinetic analysis can be performed complementing the basic 18F-FLT PET imaging would gain its final position in the diagnostic evaluation and prognostication of glioma patients.Radiolabeled FLT can serve as an in vivo marker of cell proliferation, providing valuable information regarding brain malignancies in combination with tumor proliferative biomarkers. However, further prospective cohort studies, with greater number of participants, are required before


Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans-complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D155 residue in the 30K-like movement protein-specific LxD/N50-70G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants.Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of  Capsicum annuum L.) and tomato (Solanum lycopersicum L.) crops in Queensland, Australia in 1999  is the best-studied tospovirus. Molecular biology of TSWV infection has been studied using various model plant and animal systems , 2009. TAlthough CaCV has been identified more than a decade ago, it has not been widely studied. Hence, to understand the molecular basis of CaCV infection process while constrained by the lack of a reverse genetics system for tospoviruses, we determined intracellular localization and interactions of its proteins and discuss these data in the context of viral replication, particle assembly, and intercellular movement. Furthermore, we have experimentally demonstrated the functions of CaCV non-structural proteins NSm and NSs that are involved in cell-to-cell movement and RNA silencing suppression, respectively.TM technology. Briefly, cDNA was synthesized from total RNA extracted from CaCV (QLD-3432)-infected capsicum using SuperScript III First-Strand Synthesis SuperMix (Life Technologies) and random primers. CaCV ORFs encoding N, NSs, NSm, Gc, and Gn proteins were amplified with gene-specific primers using Phusion High Fidelity polymerase (Finnzymes). To facilitate subsequent Gateway cloning, primer sequences were flanked with attB recombination sites at 5′ ends. PCR amplicons were gel-purified , cloned into pDONR221 using BP Clonase II Enzyme Mix (Life Technologies) and transformed into chemically competent Omnimax Escherichia coli (Life Technologies) using heat shock method. Recombinant colonies were selected on Luria-Bertani (LB) agar containing 50 mg/L kanamycin. Plasmid DNA was extracted from recombinant bacterial cultures using GeneJet plasmid purification kit (Thermo Fisher Scientific) and sequenced. Sequence-validated entry clones were recombined into C-series of pSITE and pSITE II destination vectors (Agrobacterium tumefaciens LBA 4404.All CaCV ORFs except that coding for RdRP were amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into plant expression destination vectors using Gateway vectors  as fusio600) were infiltrated into Nicotiana benthamiana epidermal leaf cells as previously described (N. benthamiana wild-type or RFP fused to histone 2B (RFP-H2B) transgenic marker plants, or capsicum cv. Yolo Wonder were used, whereas for BiFC protein–protein interaction assays, cyan fluorescent protein fused to histone 2B (CFP-H2B) transgenic marker plants were used  by infiltration into leaves 30 min before visualization. For BiFC assays all viral proteins were expressed as C-terminal fusions to the amino- or carboxy-terminal portions of YFP using pSITE-BiFC-nEYFP and pSITE-BiFC-cEYFP vectors  with slight modifications. Briefly, leaves from greenhouse-grown, 4-weeks old N. benthamiana were co-agroinfiltrated with pSITE constructs carrying RFP-NSm and free eGFP (PhytoTechnology Laboratories), and 4.55 g mannitol. Released protoplasts were filtered through a nylon cell strainer with 70 μm diameter pores  and collected on a 20% sucrose cushion after centrifugation for 10 min at 100 ×g. Protoplasts were collected from the interface and resuspended in 0.6 M mannitol. The suspension was centrifuged again to collect protoplasts and resuspended in mannitol solution to adjust volume and concentration. Protoplast viability was tested by staining with 1% Evan’s Blue (Sigma–Aldrich). Viable protoplasts were counted using a haemocytometer  using a light microscope with 10 × 10 magnification (Olympus BH2). Protoplast suspension was spotted on poly-L-lysine coated glass slides (Sigma–Aldrich) and incubated with constant light at room temperature. Protoplasts were observed with Zeiss LSM-700 (Carl Zeiss) CLSM starting from 4 to 8 h of incubation. Images were acquired using 40x lens as CZI files and processed using Zen Lite 2012 and ImageJ software  (1:1000) directed against NSs of watermelon silver mottle virus (WSMoV) serogroup viruses -based movement n system  derived n system . This syn system . NSm or ®Site-Directed Mutagenesis kit (New England Biolabs) following manufacturer’s instructions. Primers for mutagenesis were designed using NEBaseChanger (New England Biolabs) online following the recommendations for substitution primers. Mutations in entry clones were confirmed by sequencing. Mutants were recombined into pSITE-RFP-C1 vector and transformed into A. tumefaciens LBA 4404.Sequence-validated CaCV and TSWV NSm entry clones were used as templates to generate movement-defective NSm (ΔNSm) constructs using Q5A. tumefaciens LBA4404. Agrobacteria harboring recombinant pBIN vector carrying mGFP5-ER gene were used as inducer of RNA silencing. Agrobacteria carrying Flag-NSs and mGFP5-ER constructs were co-infiltrated with 1:1 ratio into GFP-expressing N. benthamiana line 16c  or 3 dpi (suppression of RNA silencing assay) were extracted in protein sample buffer  and sepaDeduced amino acid sequences of CaCV proteins were analyzed for potential nuclear localization (import) and nuclear export signals using cNLS Mapper  and NetNFigure 1).To determine intracellular localization of viral proteins, we transiently expressed all individual CaCV proteins except RdRP as fusions to the C-terminus of GFP and visualized fusion protein accumulation using CLSM. Expression of GFP-N and GFP-NSs fusion proteins was confirmed by western blot at 3 dpi (data not shown). Experimental evidence for expression of all GFP fusion proteins is provided by confocal microscopy showing altered intracellular localization compared to free GFP  nuclei of protoplasts expressing NSs . In contrast, we did not detect any fluorescence in the nuclei or the cytoplasm of control protoplasts or when mouse IgG was used for detection. Nuclear localization of NSs was seen in >95% of a total of 50 nuclei observed in two independent experiments based on the number of green fluorescing nuclei.We validated nuclear accumulation of CaCV NSs by immunofluorescence using a MAb directed against a common epitope in the NSs of tospovirus members of the WSMoV serogroup  in protoFigure 3). In addition mCherry-Golgi weakly labeled the ER. Both GFP:Gc and GFP:Gn appeared largely non-Golgi associated, however, some orange colored spots appeared with GFP:Gn due to co-localization of GFP and mCherry fusion proteins in merged images . Light orange colored spots were also visible with free GFP control and GFP:Gc co-localized with mCherry-Golgi. When enlarged, co-localization spots of Gn could be more clearly distinguished with distinct orange color compared to free GFP and Gc spots. Therefore, it appears that Gn, but not Gc localizes infrequently within Golgi stacks. To determine whether co-expression of both glycoproteins affects their localization, we transiently co-expressed the glycoproteins as GFP and/or Flag fusions. Co-expression of Gc and Gn did not show an altered localization profile compared to the individual fusion proteins (data not shown).We tested the potential association of individual CaCV glycoproteins with the Golgi complex by transient co-expression of GFP:Gc and/or GFP:Gn with mCherry-Golgi marker . With thFigure 1 and Supplementary Figure N. benthamiana cells transiently expressing CaCV GFP:NSm by confocal microscopy at 2 dpi relative to the location of aniline blue fluorochrome which stains callose-rich regions at PD. GFP:NSm punctate spots on the cell periphery showed perfect co-localization with aniline blue dye indicating PD localization of CaCV NSm .Based on well-studied NSm proteins of other tospoviruses, we predicted that CaCV NSm is involved in viral cell-to-cell movement. This hypothesis is supported by NSm protein localization at PD as evidenced by discontinues punctate spots in the cell periphery (S-transferase (GST) was used as non-binding control. BiFC interactions were seen as restoration of YFP fluorescence . We observed homotypic (self) interactions of N, NSs, and NSm. N–N interactions appeared as abundant aggregates in diverse sizes in the cytoplasm and perinuclear region, similar to N protein localization . No other interactions were observed by BiFC in either orientation. None of the CaCV proteins showed any interaction with GST in either orientation .We used BiFC to determine CaCV protein–protein interactions and localization in all pair-wise combinations. Individual proteins were transiently expressed in transgenic CFP-H2B plants as fusions to the C-terminus of YFP n- and c-terminal fragments. Glutathione-trans-complementation assay. NSm proteins fused to RFP (so their expression could be verified by CLSM) were transiently expressed in N. benthamiana epidermal leaf cells together with a movement-defective TVCV replicon (TVCVΔMP) composed of three modules; defective movement module, GFP reporter module, and integrase module. Complementation of TVCVΔMP movement was observed by confocal microscopy as spread of the GFP reporter replicon across five or more adjacent cells. Empty RFP vector was used as negative control and homologous P30:RFP as positive control.We provide experimental evidence that both CaCV and TSWV NSm function as cell-to-cell movement proteins in a tobamovirus replicon Figure 6A). By contrast, when TVCVΔMP was co-expressed with empty RFP vector, GFP expression was limited to single cells . These data provide evidence that tospovirus NSm was able to facilitate cell-to-cell movement of a movement-defective TVCV replicon. However, cell-to-cell movement pattern of TVCVΔMP replicon appeared different when complemented by CaCV NSm versus TSWV NSm. CaCV NSm trans-complemented TVCVΔMP movement radially in all directions generating large clusters of cells similar to TVCV P30. On the other hand, TSWV NSm trans-complementation often led to movement in a single direction generating a line of GFP-expressing cells and rarely led to radial clusters (data not shown). Efficiencies of movement trans-complementation by TVCV P30, CaCV NSm, and TSWV NSm were 65, 51, and 36%, respectively (Table 1).Co-expression of TVCVΔMP modules with CaCV or TSWV NSm showed many GFP-expressing cell clusters comprising five cells or more, similar to the TVCV P30 control at 7 dpi  constructs by replacing D (aspartic acid) residue with A  in the conserved motif. At 7 dpi, TVCVΔMP co-expressed with CaCV or TSWV RFP-ΔNSm or ΔP30-RFP showed limited GFP expression confined to majority of 1–2 isolated cells similar to vector control . Taken together these data provide strong evidence that CaCV and TSWV NSm possess cell-to-cell movement activity that was able to trans-complement movement of a dysfunctional tobamovirus replicon, and mutation of the essential D residue in the LxD/N50-70G motif specifically eliminated this movement activity.Secondary structure prediction showed tospovirus MPs contain the conserved 30K protein-specific LxD/N50-70G motif also effects PD localization, N. benthamiana leaves transiently expressing CaCV RFP-NSm or -ΔNSm were infiltrated with aniline blue to stain callose at PD. Wild-type RFP-NSm localized to the cell periphery in punctate spots  similar to GFP-NSm , whereas RFP-ΔNSm was instead smoothly distributed along the cell periphery . RFP-NSm aggregates perfectly overlapped with aniline blue spots indicating PD localization. Collectively, these data confirm that mutation of D155 residue in CaCV LxD/N50-70G motif eliminates PD localization and cell-to-cell movement activity of NSm protein, suggesting that CaCV NSm is a 30K-like viral movement protein.To determine whether substitution of D residue in LxD/NN. benthamiana leaves that transiently expressed RFP-NSm (CaCV or TSWV). TSWV NSm was used as a positive control and free GFP expression was used to highlight location of cellular structures, ER, and nucleus. Tubular structures appeared after 5 h incubation of protoplast suspensions on poly-L-lysine coated slides . Often, tubular structures looked like short projections, but occasionally longer tubules were seen. These results provide evidence that CaCV NSm, like its TSWV counterpart is capable of forming tubular structures, which may facilitate cell-to-cell movement of the virus in infected plant cells.We observed tubule-like projections on the surface of protoplasts that had been isolated from N. benthamiana line 16c, which constitutively expresses mGFP5-ER. The known suppressor TSWV NSs and pSITE-Flag vector were used as positive and negative controls, respectively. Transient expression of Flag-tagged NSs proteins was confirmed by western blot at 3 dpi (data not shown). Co-expression of CaCV NSs with GFP reporter gene enhanced visible GFP expression at 3–4 days after infiltration locally in the infiltrated leaf patch . GFP fluorescence was strong and comparable to that seen with TSWV NSs. Leaf patches infiltrated with empty vector showed significantly diminished GFP fluorescence, indicating silencing of the reporter gene. This data confirms that CaCV NSs is a local RNA silencing suppressor. GFP fluorescence in the CaCV NSs expressing patch remained bright at 7 dpi, while gfp reporter in TSWV NSs and vector leaf patches were silenced . To investigate if NSs has an effect on the short-distance spread of gfp silencing, the formation of an indicative ‘red zone’ was monitored at 7 dpi. Wide red zones were clearly visible surrounding the leaf patches expressing TSWV NSs and vector . At that time point, local GFP expression in the infiltrated leaf patch had ceased/was silenced (TSWV NSs silencing suppression was overcome), while systemic movement of the silencing signal had been initiated. By contrast, a narrow red zone was visible surrounding the leaf patch expressing CaCV NSs. This data indicates that CaCV NSs delays or interferes with the short-distance spread of the RNA silencing signal.We demonstrate that CaCV NSs functions as a suppressor of RNA silencing using well-established GFP reporter assays . CaCV NSN. benthamiana line 16c plants that had been agroinfiltrated with NSs constructs or empty vector plus mGFP5-ER. Leaves were considered systemically silenced if complete or partial chlorophyll auto fluorescence (red) was observed whereas leaves were considered non-silenced/ suppressed if GFP fluorescence resembled that of the mock (buffer-infiltrated) control plants. At 21 dpi, in two independent experiments all plants infiltrated with empty vector and mGFP5-ER constructs (16/16) showed GFP silencing in upper non-infiltrated leaves , whereas in most plants that were agroinfiltrated with constructs expressing mGFP5-ER plus CaCV NSs (15/16) or TSWV NSs (14/16) systemic silencing was suppressed and GFP expression was observed . This data indicates that CaCV NSs suppresses GFP silencing systemically with a similar efficiency as TSWV NSs.To investigate systemic silencing activity of CaCV NSs, GFP fluorescence in upper non-infiltrated leaves was monitored in Tomato spotted wilt virus is the best-studied tospovirus in terms of particle morphology, genome organization, replication and transcription strategies and therefore TSWV represents the prototype tospovirus . HoweverN. benthamiana and capsicum, suggesting that protein localization may be independent of the host plant species. N protein localization showed aggregates abundantly dispersed in the cell periphery and in the perinuclear region in association with ER. As a structural protein that binds to and protects viral genomic RNA  including TSWV, CaCV-AIT (Thailand), and INSV . Second, these NSm spots appear to coincide with PD because they perfectly co-localize with callose deposits stained with aniline blue . Third, NSm self-interacts at PD to form tubular structures that are predicted to traverse PD. Tubule-like structures composed of RFP:NSm were seen protruding from the periphery of protoplasts . Fourth, NSm functionally trans-complements a movement-defective tobamovirus replicon .We provide several lines of evidence to conclude that CaCV NSm represents the viral cell-to-cell movement protein. First, transiently expressed NSm localizes to and self-interacts on the cell periphery forming distinct punctate spots  that may ultimately facilitate virus multiplication and spread in the host plant. Furthermore, these data hint at the complex nature of viral-viral and probably viral-host protein interactions. In conclusion, the present study demonstrates that compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, protein–protein interactions, and protein functions which likely effect viral multiplication and movement in host plants.Integrated localization and interaction data or interactomes of tospoviruses studied so far show both shared and distinct properties of viral proteins (SWG and RD conceived and designed the experiments; SWG performed the experiments; SWG and RD analyzed the data; RD contributed reagents/materials/analysis tools; SWG and RD wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bamboo mosaic virus (satBaMV), the only satRNAs of the genus Potexvirus, have become one of the models of how satRNAs can modulate HV replication and virus-induced symptoms. In this review, we summarize the molecular mechanisms underlying the interaction of interfering satBaMV and BaMV. Like other satRNAs, satBaMV mimics the secondary structures of 5′- and 3′-untranslated regions (UTRs) of BaMV as a molecular pretender. However, a conserved apical hairpin stem loop (AHSL) in the 5′-UTR of satBaMV was found as the key determinant for downregulating BaMV replication. In particular, two unique nucleotides (C60 and C83) in the AHSL of satBaMVs determine the satBaMV interference ability by competing for the replication machinery. Thus, transgenic plants expressing interfering satBaMV could confer resistance to BaMV, and interfering satBaMV could be used as biological-control agent. Unlike two major anti-viral mechanisms, RNA silencing and salicylic acid-mediated immunity, our findings in plants by in vivo competition assay and RNA deep sequencing suggested replication competition is involved in this transgenic satBaMV-mediated BaMV interference. We propose how a single nucleotide of satBaMV can make a great change in BaMV pathogenicity and the underlying mechanism.Satellite RNAs (satRNAs) are sub-viral agents that may interact with their cognate helper virus (HV) and host plant synergistically and/or antagonistically. SatRNAs totally depend on the HV for replication, so satRNAs and HV usually evolve similar secondary or tertiary RNA structures that are recognized by a replication complex, although satRNAs and HV do not share an appreciable sequence homology. The satRNAs of  SatRNA mimicry is mostly conserved in higher-order RNA structures. As well, satRNAs may adopt different mimicry at different stages of virus infection such as replication and translation (Satellite RNAs (satRNAs) are short RNA molecules that share no or little sequence homology to their cognate helper virus (HV) but totally depend on the HV for replication, encapsidation and efficient movement . The homnslation .Bamboo mosaic virus (BaMV), Cucumber mosaic virus (CMV), Peanut stunt virus (PSV), Grapevine fanleaf virus, Artichoke mottled crinkle virus, Cymbidium ringspot virus (CymRSV), Tobacco ringspot virus (TobRSV), and Groundnut rosette virus (GRV)  .Satellite RNAs of BaMV (satBaMVs) are well studied. Natural isolates of satBaMVs have been collected from BaMV-infected symptomatic bamboo plants worldwide to analyze the genetic evolution and phylogeny of satBaMVs . The mimIn this review, we focus on studies of interfering satBaMVs and a possible mechanism of satBaMVs interfering in BaMV infection.Bamboo mosaic virus is a single-stranded positive-sense RNA virus containing five open reading frames (ORFs) that belongs to the genus Potexvirus of the family Alphaflexiviridae .iviridae . ORF1 eniviridae , helicasiviridae  and RNA-iviridae . ORF2 toiviridae , 2006. Oiviridae  and sympiviridae  . P20 is not essential for satBaMV replication  and all populations from Bambusa vulgaris. All satBaMVs in clades II and III are derived almost entirely from Ma bamboo from the Taipei Botanical Garden in Taiwan and B. vulgaris in India, respectively , but the secondary structures greatly differed, as revealed by enzymatic probing with RNases A, T1, T2, and V1. The secondary structures of the non-interfering BSF4 5′-UTR contain a large stem loop (LSL) and a small stem loop (SSL)  and negative (-) strands without altering satBaMV level in a plants . Moreovea plants . Furtherm quinoa . Thus, t81UGC83) in IL-1 was found in all natural interfering satBaMVs, whereas a less-conserved AHSL structure or identical AHSL structure but with different sequence (81UGU83) in IL-1 was found in non-interfering satBaMVs .On further analyzing the RNA secondary structure of natural satBaMV isolates, an identical AHSL structure was found shared by all natural interfering satBaMVs. SatBaMV mutants that swap the AHSL region of BSF4 and BSL6 revealed that the AHSL in the 5′-UTR is the determinant of satBaMV-mediated BaMV interference . To furtsatBaMVs . Furtherhenotype . AnotherMV level . Thus, b215, C286 and A330 of WLM2-satCMV could independently affect necrosis induction with different CMV strains  are essential for satBaMV-mediated BaMV interference   of satRNAs (sat-sRNAs) can target HV and induce silencing of HV for CMV ; the satRNA silencing is the major antiviral defense mechanism operating in a sequence-specific manner in plants . In geneFigure 1B), BaMV-sRNAs and satBaMV-sRNAs of BSF4 and BSL6 generated from this region are extremely low in number  of satBaMV 5′-UTR are critical for the interfering satBaMV reducing BaMV level and infection in plants. This AHSL structure and the critical nucleotide C in IL-1 is conserved in the BaMV 5′-UTR and also important for replication. Moreover, interfering satBaMV dose-dependently reduces BaMV level. Thus, interfering satBaMV-reduced BaMV level competes for the replication complex.The AHSL secondary structure and two unique nucleotides  genomic and sub-genomic RNA level without affecting (-) sub-genomic RNA level. As well, the reduced (+) genomic RNA level is greater than the (+) sub-genomic RNA level  is the most privileged. Cytosine can be easily transformed into uracil via deamination. However, m5C cannot be converted to uracil. Cellular RNAs containing m5C include transfer RNA (tRNA), ribosomal RNA, mRNA and non-coding RNA in both eukaryotes and prokaryotes - and (-)-strand level. Bisulfite sequencing  or specific satBaMV-sRNAs may likely target the host gene, which is important for BaMV replication or essential effectors of the host innate immune system other than RNA silencing. Thus, the involvement of RNA silencing in BSL6-mediated interference remains an open question. It could be evaluated by using plant mutants defective in key components of RNA silencing or plants overexpressing VSRs and further analyzing satBaMV-induced specific satBaMV-sRNAs, miRNAs and other types of host endogenous small RNAs. The mechanism underlying interfering satBaMV reducing BaMV level and host symptom development remains a fascinating question requiring long-term study.Drafting the article: K-YL, Critical revision of the article: N-SL and final approval of the version to be published: K-YL and N-SL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hematopoiesis is probably the best-understood stem cell differentiation system; hematopoietic stem cell (HSC) transplantation represents the most widely used regenerative therapy. The classical view of lineage hierarchy in hematopoiesis is built on cell type definition system by a group of cell surface markers. However, the traditional model is facing increasing challenges, as many classical cell types are proved to be heterogeneous. Recently, the developments of new technologies allow genome, transcriptome, proteome, and epigenome analysis at the single-cell level. For the first time, we can study hematopoietic system at single-cell resolution on a multi-omic scale. Here, we review recent technical advances in single-cell analysis technology, as well as their current applications. We will also discuss the impact of single-cell technologies on both basic research and clinical application in hematology. The common landscape of cellular hierarchy has been depicted as a transitional process through multiple intermediate states. In mammalian systems, many stem and progenitor cell types have been identified with a combination of cell surface markers, analyzed through multicolored fluorescence-assisted cell sorting (FACS). In hematopoietic system, extensive efforts have gone into the characterization of cellular differentiation pathways and genetic regulatory network . The ideThe signaling pathways and transcriptional networks that regulate hematopoietic stem cell emergence, self-renewal, and differentiation are not well understood , 13. PreNot until recent years, single-cell analysis has become a powerful tool for studying cellular differentiation pathways. Advances in technology made it possible to track and capture single cells, then analyze its genome, transcriptome, and proteome. For instance, high-throughput single-cell quantitative PCR is highly sensitive in detecting quantitative differences –18. SingThe cellular heterogeneity is concealed in the analysis of bulk cells. Cells gathered from the same part of the tissue differ from each other in gene expression and epigenetic status. The single-cell analysis provides a solution to understand the heterogeneity within cell population. Recently, technical developments have been made in the amplification of rare nucleic acid templates Table .Table 1CSeveral single-cell whole genome amplification (WGA) methods have been developed to amplify the rare genomic DNA. Degenerate-oligonucleotide PCR was used for analyzing copy number variation in cancer cells . AnotherSingle-cell transcriptome analysis remarkably serves as a powerful tool for studying cellular heterogeneity and lineage hierarchy Fig. . There aRecently, application of single-cell transcriptomic analysis has rapidly spread to many areas such as early embryonic development , 40–44, Traditional single-cell protein analysis depends on fluorescence flow cytometry . The devSingle-cell epigenomic technologies are becoming more and more accessible. Single-cell reduced representation bisulfite sequencing (scRRBS) and single-cell 5hmC-sequenceing were applied to investigate DNA methylation –61. SingSingle-cell capturing is a challenge. However, we have seen the significant progress of platform developments in recent years Table . When saFluidigm C1 system, a commercialized single-cell library preparation platform, uses microfluidic circuits for single-cell capture and mRNA amplification reaction . The whoDEPArray is another commercialized imaging-based cell sorting platform, which is suitable for selecting rare cells from sample. First, cells are loaded to a microfluidic cartridge, which contains an array of individually controllable electrodes. Then, every individual cell is trapped in a dielectrophoresis (DEP) cage. After imaging, cells of interest can be moved into parking chamber and recovery chamber. This technique allows isolation of tumor cells from the tissue and blood  and collThe Cyto-seq method uses a microwell array to capture a large number of single cells . IndividOne notable high-throughput platform developed very recently is the droplet-based microfluidics , which cSingle-cell imaging enables phenotypic characterization of single cells, while preserving their spatial information. DEPArray has image-based single-cell sorting function. Besides, time-lapse microscopy allowed continuous live image on single cells. This method provides important information of the dynamic cell fate decision process during blood cell differentiation , 76. HarSingle-cell transplantation is a powerful approach to verify the stem cell identity. Single-cell transplantation of hematopoietic stem cell (HSC) purified by different surface marker proved their multilineage reconstitution function , 78. RecThe next-generation sequencing platforms generate massive data set. The large amount of cell and gene dramatically increases the dimension and complexity of the data. Traditional computational methods are no longer suitable. Several single-cell computational methods have been developed. Here, we briefly introduce the workflow of single-cell data analysis.Single-cell sequencing generates huge amount of data. In single-cell transcriptomic sequencing analysis, lots of computational tools are developed for preprocessing, normalization, and transcripts quantification. A recent review gave a standard pipeline of handling single-cell RNA-seq data with or without UMIs . For sint-distributed stochastic neighbor embedding (t-SNE) [t distribution method is introduced in t-SNE to compute the similarity between two points. As a powerful visualization tool, it has demonstrated great capacity in recent high-throughput single-cell studies [The downstream analysis focuses on visualizing the high-dimensional single-cell gene expression data and clustering the transcriptionally distinct subgroups. One visualization tool for high-dimensional data is principal component analysis (PCA), which has been widely applied in single-cell research , 25, 87. (t-SNE) , 89. In  studies , 47. PCA studies . Other d studies , 91.During lineage commitment process, the continuity of single-cell gene expression can be used to infer differentiation pathway. Spanning-tree progression analysis of density-normalized events (SPADE) analysis uses this idea to infer cellular hierarchy from large-scale single-cell data set without assigning temporal order , 92, 93.Classical knowledge about hematopoiesis is built on cell type definition system using flow cytometry analysis. Such a system is limited with a small number of cell surface markers for cell classification. Recently, taking the advantage of the aforementioned technologies and the related data analysis pipelines, we start to realize that many of these classical cell types are heterogeneous. Single-cell analysis helped to provide unprecedented insights into long-lasting questions in topics of hematopoietic study, such as HSC heterogeneity, differentiation pathway, fate decision, regulatory network, HSC aging, and HSC origin.Blood cell production depends on HSC’s self-renewal and multilineage differentiation abilities. However, hematopoietic stem cells are heterogeneous in differentiation behavior. Single-cell transplantation is the most definitive assessment of HSC functional heterogeneity. Classical single-cell transplantation experiment showed that HSC defined by mouse homolog of CD34 reconstituted the lymphohematopoietic system for more than 3 months in mice. Highly purified mouse HSCs based on the expression of CD34 demonstrate variability in self-renewal potential and multilineage differentiation potential . A recenIn the classical model of hematopoiesis, an organized hematopoietic lineage tree starts with multipotent HSC, and then followed by oligopotent and unipotent progenitors. However, recent single-cell results challenged the classic model and proposed that traditional hematopoietic progenitor types are very heterogeneous , 100. GuSingle-cell mass cytometry is also a widely used method in hematopoietic differentiation pathway study. A recent primer article detailed the development of mass cytometry and related data analysis methods . BendallAltogether, these findings challenged the current step-by-step bifurcation hierarchy in hematopoietic system. Transcriptional heterogeneity among progenitors suggested that cell differentiation may proceed in a more sophisticated way. Single-cell analysis helped to rebuild hematopoietic lineage hierarchy and identify markers of previously unknown subpopulations.How do blood cells make their lineage decisions? Single-cell analysis helped to resolve this question with higher precision. Pina et al. used single-cell qPCR to define primary multipotent self-renewing cells and early erythroid-committed cells . Their dSingle-cell imaging and tracking methods are also powerful tools for examining blood generation and lineage determination. Eilken et al. used time-lapse microscopy to achieve continuous long-term single-cell observation and detected hemogenic endothelial cells giving rise to blood cells . Hoppe eThe explosion of genomic data enabled generation of regulatory networks in various biological systems. However, network build with data from population of cells are intrinsically flawed, because the fundamental unit of gene regulation is single cell, rather than heterogeneous population. Regulatory model at single-cell level starts to emerge in recent years. In 2013, Moignard et al. analyzed 18 key hematopoietic transcription factors in hundreds of blood stem cells and progenitor cells . They reIt has been a debate whether intrinsic cell changes or variations in composition contributes to the systematic HSC aging. Single-cell analysis helped to provide a deeper look into this question. Grover et al. analyzed old and young HSCs transcriptomes at the single-cell level. They identified significantly increased molecular platelet priming and functional platelet bias in the aged HSCs . They obThe origin of HSCs in mammalian systems has long been a mystery. The classical study identified the primitive type of hematopoietic stem cell in yolk sac (YS-HSC). But definitive hematopoiesis is maintained by HSC originated within the aorta-gonad-mesonephros (AGM) region of the embryo , 113. DeSingle-cell analyses have achieved remarkable advancement in recent years. Hematological researches also benefited from the rapid progress of single-cell technology. Future application of these approaches will impact the field in many aspects.One important direction for single-cell analysis is to develop cost-effective methods. The present commercialized single-cell analysis equipment is limited by both cost and throughput. More economical and convenient methods are required for broad application of high-throughput single-cell analysis. As technology goes, future throughput is expected to achieve tens of thousands of single cells per experiment, while the cost is going to drop below 0.1 USD per cell.Another orientation is the large-scale single-cell database. The explosively increasing single-cell data has raised a big challenge for scientists. Data comparison between different studies remains to be difficult. Data management becomes a burden for ordinary labs. Better computational analysis methods are needed. A large-scale database and online data analysis pipeline would be extremely helpful for the integration of various data sets collected from different tissues. An open-access platform may eventually lead to the completion of human single-cell atlas database, which might significantly impact basic research and clinical diagnosis.In the future, multi-omics will be a trend for single-cell analysis, such as profiling DNA methylome and transcriptome from the same cell simultaneously . Single-In situ single-cell sequencing methods will preserve spatial information. It should be extremely helpful for studying stem cell microenvironment. Cell niches are playing essential roles for the self-renewal and differentiation of hematopoietic stem cell in vivo. Synthetic microenvironment has been used to generate functional hematopoietic stem cells –120. In The progress of basic methodologies will accelerate the diagnosis and treatment for hematological diseases. Single-cell analysis of wild-type blood cells may offer guidance for HSC generation in vitro and improve transplantation-based therapies. Single-cell analysis of leukemia cells will help to find heterogeneity and clonal composition in cancers and guide leukemia treatments . Recentl


Heterocephalus glaber), unlike other mammals, do not conform to Gompertzian laws of age-related mortality; adults show no age-related change in mortality risk. Moreover, we observe negligible hallmarks of aging with well-maintained physiological and molecular functions, commonly altered with age in other species. We questioned whether naked mole-rats, living an order of magnitude longer than laboratory mice, exhibit different plasma metabolite profiles, which could then highlight novel mechanisms or targets involved in disease and longevity. Using a comprehensive, unbiased metabolomics screen, we observe striking inter-species differences in amino acid, peptide, and lipid metabolites. Low circulating levels of specific amino acids, particularly those linked to the methionine pathway, resemble those observed during the fasting period at late torpor in hibernating ground squirrels and those seen in longer-lived methionine-restricted rats. These data also concur with metabolome reports on long-lived mutant mice, including the Ames dwarf mice and calorically restricted mice, as well as fruit flies, and even show similarities to circulating metabolite differences observed in young human adults when compared to older humans. During evolution, some of these beneficial nutrient/stress response pathways may have been positively selected in the naked mole-rat. These observations suggest that interventions that modify the aging metabolomic profile to a more youthful one may enable people to lead healthier and longer lives.Mouse-sized naked mole-rats (The online version of this article (10.1007/s11357-018-0014-2) contains supplementary material, which is available to authorized users. The comprehensive evaluation of plasma metabolites has the potential to elucidate biomarkers and metabolomic signatures associated with aging  metabolomics studies  is an excellent example of an organism that has naturally achieved both an extended health and life span (Buffenstein The mouse-sized (35–50 g) naked mole-rat  and genetic approaches, though candidate mechanism for causality could potentially be revealed through careful genomic annotation. In addition, using more global unbiased analyses (e.g. “-omics” platforms) in an experimental design that contrasts the long-lived naked mole-rat with a comparable short-lived mammal may be particularly useful for identifying distinctive characteristics of the naked mole-rat pertinent to attenuation of the aging process. Specifically, a metabolomics profile may reveal, in an unbiased way, metabolites that prove to be biomarkers of age-related survivorship and may ultimately elucidate universal mechanisms involved in slow and successful aging phenotypes. Here, we compare the metabolomic signatures of the naked mole-rat and the shorter-lived, similarly sized, and well-characterized C57Bl/6 mouse. Furthermore, using publically available data, we also compare the circulating metabolome of naked mole-rats to both fasting, metabolically suppressed hibernating squirrels . The naked mole-rat is also coprophagic consuming fecal pellets that contain considerable amounts of the microfauna found in their large cecum  and five C57BL6/J male mice (6 months old) were used in this study. The naked mole-rats were part of the well-characterized Buffenstein colony, and the mice were purchased from the Jackson Laboratories  and maintained in the vivarium for at least 1 month prior to use. The ages selected yielded young, healthy individuals that were physiologically age-matched (~ 15–20% of their observed maximum life span). Both species were maintained on a 12-h light-dark cycle. Naked mole-rats were housed in family groups in interconnected systems consisting of tubes and cages of varying sizes to simulate the multi-chambered burrow and tunnel systems that the species inhabits in the wild. Climatic conditions also approximated those found in their native habitat , although atmospheric oxygen was ~ 21%. Naked mole-rats met all their nutrient and water needs through an ad libitum supply of fruit and vegetables . Although the staple diet of the naked mole-rat (yams) has approximately 10-fold lower levels of total protein than mouse chow  and C57BL6/J mice (n = 5) were used in this study. All animals used in this study were sacrificed in the late morning (10.30am -12 noon) after having access to fresh food. Twenty-four hours prior to anesthesia with isoflourane, all animals received an oral 200-μl bolus of sesame oil to serve as control animals for other ongoing studies, and were then sacrificed by cardiac exsanguination. The blood was collected in the presence of EDTA, mixed by inversion and incubated on ice (maximum of 30 min) before centrifugation . Plasma samples (~ 0.5–1.5 ml) were collected and stored (− 80 °C) until transfer on dry ice to Metabolon Inc. , where they were further stored (− 80 °C) without thawing until analysis.Physiologically age-matched, young male naked mole-rats (www.metabolon.com) whose platform and protocol have been described in detail elsewhere  correction procedure (BH procedure)  we performed an enrichment analysis using the R Bioconductor fgsea package  values. The major metabolite clusters were visually determined according the dendrogram topology. To test for enrichment of super- and sub-pathway terms (assigned to each identified metabolite by Metabolon Inc.) in each of the metabolite clusters, we performed a hypergeometric test per each super- and sub-pathway term using the R Bioconductor piano package  and the late torpor versus torpor entrance mean abundance ln(fold change) in ground squirrels, we filtered the 74 intersecting metabolites, keeping metabolites with low noise (computed as the coefficient of variation: error/mean of the estimated ln(fold change)) associated with their ln(fold change) estimate since metabolites with noisy ln(fold change) estimates can be randomly distributed around zero and therefore may mask any possible association  and that of the methionine-restricted versus control-fed rat diet mean abundance ln(fold change) in rats. To do so, we filtered the 213 intersecting metabolites keeping strongly differentially abundant ones; for similar reasons, we applied filtering in the analysis with the 13-lined ground squirrel data , indicating no species-specific systematic biases in our approach.In order to identify metabolites whose abundances may be associated with the different life spans of the naked mole-rat versus the C57Bl/6 laboratory mouse, we profiled plasma metabolites from nine adult naked mole-rats and five adult mice using the Metabolon Inc  platform (see Methods). We obtained abundance measurements for 575 metabolites, 360 of which were identified  and in the fibrinogen cleavage sub-pathway nested within the peptide super-pathway . In addition, a significant enrichment of the lysophospholipid sub-pathway  was found in the other naked mole-rat downregulated cluster  ; a peptide sub-pathway ; and a lipid sub-pathway . It also revealed that metabolites that are strongly upregulated in naked mole-rat relative to mouse are significantly enriched in several lipid sub-pathways involved in membrane composition, sterols, and secondary bile metabolism  . Similarly, the long-lived insulin receptor substrate 1 null (Irs−/−) mice also have very low circulating levels of methionine. In contrast, long-lived Ames dwarf mice reportedly have increased plasma methionine levels relative to control mice, though levels of other amino acid metabolites were more in keeping with the naked mole-rat. Tryptophan levels in naked mole-rats were also ~ 55% those of mice , and naked mole-rat levels of serotonin, of which tryptophan is the precursor, were very low (~ 6.5%) relative to those in mice . In contrast, levels of degradation products of tryptophan were all higher in naked mole-rats than in mice . Similar to tyrosine, the levels of the aromatic amino acid phenylalanine were also lower in naked mole-rats compared to mice , while again the levels of many of phenylalanine’s degradation products  were higher in naked mole-rats . These data suggest that when compared to mice, naked mole-rats show greater levels of amino acid degradation. Notably, circulating levels of both branched-chain amino acids  and aromatic amino acids have been positively correlated with insulin resistance and implicated in diabetes  show increases in pools of branched-chain amino acids compared to wild-type worms . Glutamate is functionally critical in glutathione (GSH) synthesis, an important signaling molecule and also a major excitatory neurotransmitter, and its product, GABA, as a key inhibitory neurotransmitter . GGAA synthesis is catalyzed by gamma-glutamyltranspeptidase (GGT) and involves the transfer of the glutathione (GSH) glutamyl moiety to free amino acids to form GGAAs. These are then transported across the cell membrane and converted by intracellular gamma-glutamylcyclotransferase to the corresponding amino acids and 5-oxoproline. This process is dependent upon the availability of GSH . Such low levels likely reflect the naked mole-rat’s strict herbivorous diet and concomitant low creatine levels in the fruit and vegetables it consumes. Low levels of this organic acid may also reflect the lower basal metabolic rate of the naked mole-rat, 66–75% of that of mice  palmitoyl sphingomyelin (d18:1/16:0) and stearoyl sphingomyelin (d18:1/18:0) levels, respectively, compared to mice. In addition, most of the identified medium- and long-chain fatty acids, as well as sterols, were also found to have significantly higher levels in the naked mole-rats relative to mice; notable exceptions were nonadecanoate (19:0), docosahexaenoate , as well as the essential fatty acid linoleate (18:2n6), which were lower in naked mole-rats relative to mice. Linoleate is commonly converted into arachidonate and other PUFAs that are elevated in the naked mole-rats. The increase in PUFAs was associated with a significant drop in lysophospholipids and an increase in sphingolipid metabolism.In addition to the amino acid and peptide super-pathways, the lipid super-pathway was found to be enriched in metabolites with a strong species difference; in many cases, with higher levels in naked mole-rat sub-pathways relative to mice. These included several fatty acid metabolites as well as components of bile metabolism. A similar, albeit-graded enrichment of lipid-associated metabolites was observed in the livers of young mice subjected to different degrees of caloric restriction where several lipid metabolites were increasingly upregulated with increasing severity of caloric restriction  in the naked mole-rats may reflect their disparate membrane composition and greater membrane stability . Sphingomyelins are synthesized from phosphorylcholine, sphingosine, and an acylated group, such as a fatty acid. Palmitoyl sphingomyelin is a form of sphingomyelin containing palmitate (16:0) at the variable acylation position. Palmitoyl sphingomyelin interacts with cholesterol in ordered lipid rafts in cell membranes, whereas stearoyl sphingomyelin is also a major constituent of membranes interacting with cholesterol and is a key component of vesicular trafficking . Amphipathic sphingolipids are not only a major constituent of cell membranes, in particular the myelin sheath of nerves, but are also a major constituent of biologically active molecules controlling many cellular processes, including cell division, differentiation, apoptosis, and signaling  and stearoyl sphingomyelin (d18:1/18:0) were, respectively, 15- and 11-fold higher levels in naked mole-rats . As an example, cholesterol levels were ~ 2.5-fold higher in naked mole-rats. Although cholesterol levels are strongly positively associated with cardiovascular disease in humans  Collaboration P values = 0.1 and 0.03, respectively). In contrast, naked mole-rats showed significantly lower plasma levels of corticosterone  and higher levels of cortisol  than in mice. This confirms previous observations using ELISA and RIA assays ; glycodeoxycholate ; and ursodeoxycholate . These bile salts are primarily formed through microbial metabolism of cholic acid although ursodeoxycholate may also be formed in the liver. Such high levels of secondary bile metabolites are usually associated with a diet low in fiber and high in fat; this certainly is not the case for the naked mole-rat.Bile salts are also derived from cholesterol and play key roles in emulsifying lipid aggregates and in facilitating transport of these hydrophobic lipids in the predominantly hydrophilic plasma. Large species differences in circulating levels of bile salts were also evident, with the exception of 12-dehydrocholate and tauroursodeoxycholate. Three of these bile salts had close to an order of magnitude or higher concentrations in naked mole-rats relative to mice, namely deoxycholate  in the serum reportedly are indicative of liver disease. They are often considered both cytotoxic and carcinogenic, thereby implicated in the pathogenesis of gastrointestinal neoplasia . While it is conceivable that these unidentified metabolites may reflect novel mechanisms linked to species longevity, the possibility that they are related to species differences in the microfauna and flora or environmental substances cannot be excluded. Even if so, these differences in microbial secretions and xenobiotics may also contribute to the sustained good health and exceptional longevity of the naked mole-rat.Notwithstanding the identity of many significantly differentially abundant metabolites, some with staggering fold changes cannot be currently identified   Fig. a, mostly−3) Fig. a.Fig. 4AP value = 0.17) , hence additionally highlighting shared traits that may be directly pertinent to extreme longevity. This positive relationship is mainly driven by downregulated metabolites in the amino acid super-pathway and upregulated metabolites in the lipid super-pathway, in both studies. For example, both methionine-restricted rats and naked mole-rats show low circulating levels of pyruvate, succinate, and fumarate relative to control-fed rats and mice, respectively, in contrast to citrate which shows the opposite trend. These differences may reflect the lower metabolic rate and concomitant declines in cellular respiration.Similar to the 13-lined ground squirrel study, a metabolomics study contrasting rats on a methionine-restricted diet with control-fed rats reported significant differential abundances in metabolites in the amino acid, peptide, and lipid super-pathways  Fig. b, which Using metabolomic screens, we observed that young, healthy naked mole-rats and mice exhibit markedly distinct plasma metabolite profiles, with the most pronounced differences evident among circulating amino acid, peptide, and lipid metabolites. Moreover, the largest differences in metabolite levels were observed in several as-yet unidentified metabolites. Although the biological significance of these differences in circulating metabolites is difficult to discern, the differential findings appear to align with similar trends observed in several rodent models of extended longevity. Conspicuous differences observed between naked mole-rats and mice are evident in the methionine pathway, with plasma levels of methionine 2.8-fold greater in mice than in naked mole-rats and similar low levels of downstream metabolites of methionine metabolism Fig. . The OreThese metabolite abundance differences observed between naked mole-rats and mice were found to resemble that of hibernating ground squirrels and rats subjected to dietary/amino acid restriction. All these rodents possess several traits in common such as lower body temperatures, low thyroid metabolism, low insulin levels, and reduced metabolic rates  are lower than in older adults Supplementary Table S1(CSV 205 kb)Supplementary Table S2(CSV 84 kb)Supplementary Table S3(CSV 18 kb)Supplementary Table S4(CSV 7 kb)


Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction.In the presence of sufficient iron, the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies.We cloned the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant.The vectors pFCF1, pFCF2, and pFBH1 have been shown to use the The online version of this article (doi:10.1186/s12896-016-0298-1) contains supplementary material, which is available to authorized users. Escherichia coli, one of the major regulators of iron homeostasis is the protein Fur (Ferric Uptake Regulator), a homodimeric protein with 17 kDa subunits , 19fepB entA, entE, and entB) under the control of Fur box 2 were prepared. Chromosomal expression of these ORFs in E. coli is under Fur control, and they were therefore logical candidates for testing the plasmid-borne bidirectional promoter region in pFCF1/2 and pFBH1. CAS assays and growth studies , as well as studies on proteins that are involved in Fur-controlled virulence mechanisms  [Our current understanding of Fur regulation indicates that there are approximately 80 l of Fur . By tran system) . Further


Few effective treatment options are available for patients with advanced or metastatic urothelial carcinoma (UC) after unsuccessful first-line platinum-based chemotherapy. To date, immune checkpoint inhibitors are novel therapeutic agents for UC treatment. From May 2016 to May 2017, five anti-PD-1/PD-L1 monoclonal antibodies received accelerated or regular approval from the US Food and Drug Administration (FDA) for the treatment of patients with locally advanced or metastatic UC. The present comprehensive review presents the background information of these five US FDA-approved anticancer agents to provide a basic but concise understanding of these agents for advanced studies. We summarize their immune checkpoint mechanisms, clinical efficacy, recommended usage protocols, adverse events, and the limitations of the PD-L1 biomarker assays. Urothelial carcinoma (UC) is one of the top ten leading causes of cancer death worldwide. UC tumorigenesis is thought to be associated with environmental carcinogenic exposure such as cigarette smoking and chemical exposure . The patMycobacterium bovis, has been used for treatment in patients with nonmuscle invasive bladder UC since the 1990s. The benefits from intravesical BCG instillation have been proven, including lowering the risk of disease recurrence and disease progression , 39n = 2In addition, immune-targeted agents that can cause dysimmune toxicities in any tissue but mainly affect the lung, liver, gut, endocrine glands, and skin caused immune-related adverse events (IRAEs) . AlthougAlthough upper tract urothelial carcinoma (UTUC) was identified with molecular profiling approaches that were different from those for bladder UC , the immAccording to our review of the relevant literature, previous studies did not provide the nucleotide sequence or protein compositions of PD-1/PD-L1 immune checkpoints in patients with UC. The relationships between the antigen-binding sites (paratopes) on the therapeutic monoclonal antibody inhibitors and the antibody-binding sites (epitopes) on the checkpoint proteins remain unclear. Nonetheless, UC has been identified as the tumor with high and heterogeneous mutation burden . The genCurrently, PD-L1 is regarded as a biomarker in PD-1/PD-L1 inhibitor trials , 54–56 tAdditionally, these PD-1/PD-L1 inhibitors may exert synergistic effects with other anticancer agents to prolong patients' survival or reduce side effects. In this compact but comprehensive review, we summarized the background information of the five US FDA approved PD-1 and PD-L1 checkpoint inhibitors as well as elucidate their mechanism of actions (MOA). We outlined their drug efficacy, safety, and adverse events from the clinical trials of patients with UC. These therapeutic antibodies have shown promising results in their respective FDA-approved trials and have given new hope to those who are suffering from advanced or metastatic UC. Further large-scale clinical trials of checkpoint inhibitor will reveal the optimal administration of these drugs and allow more patients with UC to benefit from immunotherapy treatments.


Middle East Respiratory syndrome (MERS) first emerged in Saudi Arabia in 2012 and remains a global health concern. The objective of this study was to compare the clinical features and risk factors for adverse outcome in patients with RT-PCR confirmed MERS and in those with acute respiratory disease who were MERS-CoV negative, presenting to the King Fahad Medical City (KFMC) in Riyadh between October 2012 and May 2014. The demographics, clinical and laboratory characteristics and clinical outcomes of patients with RT-PCR confirmed MERS-CoV infection was compared with those testing negative MERS-CoV PCR. Health care workers (HCW) with MERS were compared with MERS patients who were not health care workers. One hundred and fifty nine patients were eligible for inclusion. Forty eight tested positive for MERS CoV, 44 (92%) being hospital acquired infections and 23 were HCW. There were 111 MERS-CoV negative patients with acute respiratory illnesses included in this study as “negative controls”. Patient with confirmed MERS-CoV infection were not clinically distinguishable from those with negative MERS-CoV RT-PCR results although diarrhoea was commoner in MERS patients. A high level of suspicion in initiating laboratory tests for MERS-CoV is therefore indicated. Variables associated with adverse outcome were older age and diabetes as a co-morbid illness. Interestingly, co-morbid illnesses other than diabetes were not significantly associated with poor outcome. Health care workers with MERS had a markedly better clinical outcome compared to non HCW MERS patients. MERS coronavirus (MERS-CoV) was initially identified in a 60-year-old male with severe progressive fatal pneumonia in Jeddah, Saudi Arabia in June 2012 ,4. HowevEarlier studies have attempted to describe the clinical features of MERS as well as compare it with SARS , 10, 11.To address these gaps in knowledge, in this study we sought to analyse the demographic characteristics, clinical data and outcomes of all patients meeting the case definition for MERS at a major hospital in Riyadh between 2012 and 2014 who were tested by RT-PCR for MERS-CoV. The clinical features and outcomes of those virologically confirmed to be MERS-CoV positive and negative were compared. In addition, we compared MERS-CoV positive HCW with MERS-CoV positive non-HCWs. The molecular epidemiology of a subset of these patients has been previously reported .The study was conducted at King Fahad Medical Centre (KFMC), a 1200-bed tertiary care hospital in Riyadh which comprises of four hospitals ; the main hospital houses four affiliated centres  in addition to the Medical and Surgical Specialty departments within the same campus. The intensive care unit (ICU) is located in the main hospital. The medical city serves as a tertiary referral centre for patients from throughout the Kingdom.Included in this study are patients who met the clinical case definition for MERS and were tested by RT-PCR for MERS-CoV RNA between October 2012 and May 2014. A confirmed case of MERS was defined by the Ministry of Health, Saudi Arabia as a febrile patient with ARI who tested positive for MERS-CoV infection. Patients with community- or nosocomially-acquired MERS as well as HCW diagnosed with MERS at KFMC were included. Patients meeting the clinical case definition during the same time frame who were MERS-CoV negative served as controls. Nasopharyngeal swabs or lower respiratory tract specimens  were collected from patients meeting the clinical case definition of MERS and tested for presence of MERS-CoV RNA.From October 2012 to April 2014, samples were tested for MERS-CoV at the MOH laboratories. At the end of April 2014 when in-house MERS-CoV testing capability was developed, all samples were tested at KFMC. RT-PCR diagnosis for MERS-CoV was based on WHO recommendations, testing the upstream and using Orf1a for confirmation [Patient demographic and clinical data were retrieved from electronic health records and via retrospective chart review.We compared various characteristics among MERS-CoV positive HCW, MERS-CoV positive non-HCW, and MERS-CoV negative, cases, by Fisher’s exact test for categorical factors or by ANOVA for continuous factors. We looked at factors associated with poor outcome (admission to ICU or death) among MERS patients, and compared the factors between patients with poor versus favourable outcome by Fisher’s exact test or ANOVA. In addition, we looked at the factors associated with death in MERS patients by fitting a logistic regression. We investigated time from hospital admission to death among subgroups, by fitting Kaplan-Meier curves, and compared subgroups by the log-rank test.The study was approved by the King Fahad Medical City Institutional Review board. Given that this was an outbreak situation they waived the requirement for individual informed consent.The demographic characteristics of 159 eligible patients were reviewed. Forty eight of the patients were confirmed to be MERS-CoV RT-PCR positive while 111 were negative in MERS-CoV RT-PCR tests . Of the A history of exposure to animals  was more common in the MERS-CoV positive patients overall, compared with MERS-CoV negative patients (p = 0.04) . A histoThe presenting clinical features and co-morbidities of MERS-CoV positive (HCW and non-HCW) and MERS-CoV negative patients are shown in The biochemical and haematologic parameters between the three groups were similar, except for higher white blood count (WBC) values in MERS-CoV negative patients and higher serum creatinine levels in MERS-CoV positive non-HCW compared with MERS negative patients . There wAmong the 48 patients with confirmed MERS, 47 had chest radiography available for review. Of these, 12 (25%) had normal chest radiographs at initial presentation, 21 (45%) had unilateral lung infiltrates and 14 (30%) had bilateral lung infiltrates. Seven (15%) of the total cohort never developed an infiltrate. Amongst those with normal initial radiographs, 3 (25%) and 2(17%) deteriorated and developed unilateral and bilateral infiltrates, respectively. Amongst patients with baseline unilateral infiltrates, 15 (71%) progressed and developed bilateral infiltrates; 4 (19%) had no changes. Two patients with bilateral infiltrates survived and had their lungs revert to normalcy.Adverse clinical outcomes such as ICU admission or death were not significantly different between MERS-CoV positive (HCW and non-HCW considered together) and MERS-CoV negative patients. However, of the MERS-CoV positive HCW, only 4 of 23 (17%) required ICU admission and 1 of 23 (4.35%) died in contrast with 21 of 25 (84%) (p<0.001) and 16 of 25 (60%) (p<0.001) of the non-HCWs with MERS-CoV infection, respectively. Furthermore, non-HCW MERS-CoV positive patients were more likely to be admitted to ICU  than MERS-CoV negative patients (62 of 111 (56%) (p = 0.02); and were more likely to die  than MERS-CoV negative patients  (p<0.001) .Among the patients with MERS-CoV infection, a poor clinical outcome (ICU admission or death) was significantly associated with older age, presence of co-morbid conditions, lower lymphocyte count, higher neutrophil count, higher serum creatinine and higher serum LDH levels, by univariate analysis . DiabeteUsing Kaplan-Meier analysis, survival was shown to be significantly different in patients with MERS-CoV infection (taken together) in comparison with MERS-CoV negative patients .Clinical, demographic and outcome data from 159 patients investigated as suspected MERS were analysed; of these 48 were virologically confirmed as MERS-CoV infected. MERS patients who were not HCW were older than HCW and those who were MERS-CoV negative. The presenting clinical features of patients with MERS were indistinguishable from those who were investigated for MERS but found MERS-CoV negative, with the exception that diarrhoea was more common in patients with MERS. This appears to be primarily because 11 of 23 (48%) HCW with MERS reported diarrhoea significantly more often than patients who were MERS-CoV negative  (p = 0006). However, there was no difference in the frequency of diarrhoea in non-HCW MERS patients and non-MERS patients. Notably, diarrhoea was also a prominent feature of SARS with evidence of virus replication in intestinal epithelium  and thusPatients with MERS who were non-HCW were more likely to > 50 years of age when compared to non-MERS patients (p = 0.0034) and MERS patients who were HCW (p = 0.0005). Patients with MERS who were non-HCW had significantly worse clinical outcomes than those who were MERS-CoV negative or HCW with MERS-CoV infection. This may be related, in part, to the fact that both HCW and non-MERS patient groups were younger and had fewer co-morbid conditions. In a previous report of MERS-CoV infection in HCW detected by active case finding of contacts, seven infected HCW were documented, two of whom were asymptomatic, while the other five had mild upper respiratory tract symptoms . The ageTaking all MERS patients together, the variables associated with poor clinical outcomes in univariate analysis were older age, presence of co-morbid illness, neutrophilia, lower lymphocyte counts, higher serum creatinine levels and higher serum LDH levels. On multivariate regression analysis, only occupation (HCW) and younger age were significantly associated with favourable clinical outcome. In survival analysis, MERS patients with diabetes (with or without other co-morbidites) had worse survival than MERS patients with other co-morbidities (other than diabetes) or those with no co-morbidities. Interestingly, patients with co-morbidities other than diabetes were not significantly different to those with no co-morbidities.Limitations of the study include the possibility that some patients with MERS-CoV may have been missed because of false negative RT-PCR results and may be categorised as non-MERS-CoV patients. Convalescent sera were not available for testing from all patients under investigation. Secondly, the data collection was retrospective. Viral load data was not available for analysis. The MERS negative cases are likely to be heterogeneous in their diagnosis. Final discharge diagnosis of this control group is unfortunately not available.In conclusion, MERS could not be reliably distinguished from patients who were MERS negative by clinical, demographic or epidemiological criteria; this highlights the need for a high index of suspicion, early isolation and early laboratory testing to identify infected patients and prevent nosocomial spread. An important observation from this study is that HCW with MERS had a markedly more favourable clinical outcome compared with MERS patients who were non-HCW, primarily because they were younger and had less co-morbidities. Clinical progression, presentation and outcomes are very different in these two groups and this has to be kept in mind and adjusted for when therapeutic trials for MERS are designed. Diabetes, rather than other co-morbidities had a significant adverse impact on survival.