Two-dimensional IR spectroscopy can be designed to eliminate the diagonal peaks and expose only the crosspeaks needed for structure determination

The power of two-dimensional (2D) IR spectroscopy as a structural method with unprecedented time resolution is greatly improved by the introduction of IR polarization conditions that completely eliminate diagonal peaks from the spectra and leave only the crosspeaks needed for structure determination. This approach represents a key step forward in the applications of 2D IR to proteins, peptides, and other complex molecules where crosspeaks are often obscured by diagonal peaks. The technique is verified on the model compound 1,3-cyclohexanedione and subsequently used to clarify the distribution of structures that the acetylproline-NH(2) dipeptide adopts in chloroform. In both cases, crosspeaks are revealed that were not observed before, which, in the case of the dipeptide, has led to additional information about the structure of the amino group end of the peptide.     

Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUB_GEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUB_GEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUB_GEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

The Ras homologous (Rho) family of GTPases is part of the Ras superfamily of small G proteins. Rho family GTPases are molecular switches that control intracellular actin dynamics and regulate a diverse array of cellular processes from cytokinesis to cell migration and wound healing (1–3). These small ∼21-kDa proteins are highly conserved in all eukaryotes, with three founding family members that have been extensively studied: Rac1, Cdc42, and RhoA. Each Rho family GTPase exerts specific effects on the actin cytoskeleton, and constitutive activation of each protein leads to characteristic cellular phenotypes.The signaling activity of a GTPase is controlled by its bound nucleotide. When GDP is bound, the GTPase is in the “inactive” state, and loading of a GTP promotes the “active” conformation of the G protein. Interaction with downstream effector proteins and subsequent actin reorganization only occurs when the GTPase is in the GTP-bound active state. The intrinsic nucleotide exchange (GDP to GTP) and hydrolysis (GTP to GDP) rates of Rho family GTPases alone are slow. Nucleotide exchange occurs on the order of 1.5 per hour (4, 5), and the intrinsic hydrolysis rate is ∼0.15 per min (6). Rapid regulation of GTPases, therefore, is controlled by two classes of proteins that either switch them “on” or “off”: guanine nucleotide exchange factors (GEFs) promote the dissociation of GDP and allow loading with GTP, and GTPase activating proteins (GAPs) accelerate the intrinsic GTP hydrolysis by the G protein.Bacterial pathogens such as certain species of Salmonella, Shigella and enteropathic Escherichia coli, encode and secrete effector proteins that modulate small GTPases to benefit the bacterium during infection. For instance, the Shigella flexineri IpgB2 GEF protein activates RhoA and causes characteristic membrane ruffles that are critical for Shigella invasion of the host cell (7). Bacterial effector GEFs belong to either the WxxxE family (named for a conserved motif important for folding and structural integrity) or the SopE family (SopE, SopE2, and BopE). These bacterial effectors share no sequence or structural homology to eukaryotic Rho GEFs, which predominantly belong to the Dbl homology (DH) family of GEFs that adopt a six-helix bundle with an elongated, kinked “chaise lounge” fold (8, 9). Rather, bacterial effector GEFs adopt a characteristic compact V-shaped fold, yet activate the Rho GTPases via the same contact regions in the GTPases that are crucial for nucleotide exchange by DH-family GEFs (10). While substantial effort has been exerted in detailing the molecular determinants of bacterial GEF activities and specificities, no bacterial effector GEFs have been identified outside of the WxxxE or SopE-like families.Recently, we identified and characterized a putative effector protein, OtDUB, from the obligate intracellular bacterium that causes scrub typhus, Orientia tsutsugamushi. Despite extensive characterization of the OtDUB deubiquitylase (DUB) domain (residues 1–259), the function of the extensive C-terminal region, encompassing more than 1,000 amino acids, remained elusive (11). Here, we report that OtDUB encodes a GEF domain, OtDUB_GEF. Using biochemical, structural, and cellular methods, we demonstrate that OtDUB_GEF predominantly activates Rac1 in vitro and in cell culture. While the primary sequence of OtDUB_GEF is unrelated to WxxxE or SopE GEFs, the OtDUB_GEF crystal structure reveals a similar V-shaped fold despite an entirely different topological and helical arrangement, suggesting convergent evolution. We further determined the OtDUB_GEF:Rac1 complex crystal structure and demonstrate that OtDUB_GEF interacts with Rac1 at key common loci in the GTPase. The complex structure also suggested a distinct mechanism for GDP displacement, unique among all GEFs characterized to date. Our work reveals that O. tsutsugamushi has evolved a GEF domain that expands the molecular repertoire of bacterial effectors and suggests a critical function for OtDUB in regulating Rac1 to benefit the pathogen during infection.     

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes

Priming of CD8⁺ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1⁺ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14⁺ CD169⁺ monocytes and Axl⁺ CD169⁺ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169⁺ moDCs and Axl⁺ CD169⁺ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8⁺ T cells. Finally, Axl⁺ CD169⁺ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169⁺ DCs to drive antitumor T cell responses.

The major breakthrough of immune-checkpoint inhibitors, such as anti-CTLA4 and anti–PD-L1, in cancer therapy is still limited to a minority of patients who respond to this treatment (1). Patients with pancreatic cancer, for example, failed to respond to monotherapies of checkpoint inhibitors in multiple trials (2, 3). Factors such as poor tumor immunogenicity, tumor-immunosuppressive microenvironment, and the lack of an existing tumor-specific immune response are thought to contribute to patients’ lack of response to these immune-checkpoint inhibitors (2, 4, 5). Nevertheless, the abundance of intratumoral CD8⁺ T cells is associated with longer survival of pancreatic cancer patients, suggesting these patients may benefit from a better antitumor immunity (6–8). Therefore, new strategies aiming to boost patients’ antitumor CD8⁺ T cell responses should be explored to improve current therapies.Dendritic cells (DCs) play a crucial role in eliciting immune responses against tumor-specific antigens and have therefore generated significant interest as a therapeutic target in the context of cancer immunotherapy (9). The most commonly used DC-based immunotherapy utilizes monocyte-derived DCs (moDCs) due to the large numbers that can be generated ex vivo. In general, moDC-based vaccines have shown some survival benefit and appear to be well-tolerated; however, the objective response rate in most studies is still relatively low (9, 10). Moreover, since generating DCs ex vivo is a laborious, time-consuming, and costly process, research is shifting toward targeting tumor antigens to naturally circulating or tissue-resident DCs in vivo as a vaccine strategy to induce immune responses (11). Both in mice and humans, DCs can be divided into several subsets, of which the conventional DCs (CD141⁺ cDC1 and CD1c⁺ cDC2) have been shown to be responsible for T cell priming (12, 13).In vivo DC targeting can be achieved by using antibodies or ligands that bind to DC-specific receptors and are directly conjugated to tumor antigen or to nanoparticles harboring tumor antigen. Targeting C-type lectin receptors in particular, such as DEC-205, Clec-9A, and DC-SIGN, has been demonstrated to induce antigen-specific and antitumor responses in mouse and human models (14–17). Recently, we compared two vaccination strategies of antigen–antibody conjugates directed to either DEC-205⁺ DCs or to CD169⁺ macrophages, a type of macrophage that acts as sentinel in secondary lymphoid organs (18). Remarkably, we observed that antigen targeting toward CD169⁺ macrophages led to a significant antigen-specific CD8⁺ T cell response that was as efficient as DEC-205 targeting and capable of suppressing tumor cell outgrowth (18–20). Stimulation of antigen-specific immune responses by targeting to CD169 has also been demonstrated using HLA-A2.1 transgenic mice and human CD169-expressing moDCs (21), indicating the immunotherapy potential of antigen targeting to CD169.In a resting state, CD169/Siglec-1 is highly expressed by a specific subtype of macrophages that are located bordering the marginal zone in the spleen and the subcapsular sinus of lymph nodes (22, 23). Their strategic location allows them to be among the first cells to encounter and to capture blood and lymph-borne pathogens, and, in conjunction with DCs, to initiate the appropriate immune responses (18, 19, 24, 25). In addition to combating infection, CD169⁺ macrophages have been implicated in antitumor immunity. They have been shown to capture tumor-derived materials in mouse and human (26, 27), and their frequency in tumor-draining lymph nodes is clearly associated with better clinical outcomes in several types of cancer (28–30). Although the exact mechanism is unclear, these observations suggest that lymphoid-resident CD169⁺ macrophages can positively contribute to antitumor immunity. Next to lymphoid tissue-resident macrophages, CD169 is also constitutively expressed by a recently described Axl⁺ Siglec6⁺ DC subset (Axl⁺ DCs, AS DCs, or pre-DCs) present in peripheral blood and lymphoid tissues (31–34). Axl⁺ DCs have been proposed as a distinct DC subset that has the capacity to produce inflammatory cytokines and to stimulate CD4⁺ and CD8⁺ T cells (31–33). In addition to these constitutively CD169-expressing macrophages and DCs, during inflammatory conditions, monocytes can up-regulate CD169 in response to type I interferons (IFN-Is) (35, 36).CD169 is a member of the sialic acid-binding Ig-like lectin (Siglec) receptor family that recognizes sialic acids present on glycoproteins or glycolipids on the cell surface and mediates cell–cell interactions and adhesion (37). Sialic acid-containing glycosphingolipids, such as GM3, GT1b, and GD1a gangliosides, are known to be endogenous ligands for CD169 molecules (38, 39). However, the CD169–sialic acid axis can be hijacked as a receptor entry molecule by viral pathogens, including murine leukemia virus (MLV), HIV, and Ebola virus to infect DCs or macrophages (40–43). The CD169-mediated entry and transinfection is dependent on gangliosides, including GM3, that are present on the viral lipid membrane (40, 44, 45). Interestingly, Axl⁺ DCs have been recently demonstrated to be the predominant DC subset to capture HIV in a CD169-dependent manner.In this study, we aimed to exploit ganglioside–CD169 interactions to develop a novel tumor antigen vaccination strategy that directs tumor antigens to human CD169⁺ antigen-presenting cells (APCs) using liposomes containing gangliosides. We generated liposomes with different types of gangliosides and assessed the binding and uptake by different types of human CD169⁺ APCs, including monocytes and primary and monocyte-derived macrophages and DCs. High-dimensionality mapping revealed the specificity of ganglioside-liposome targeting exclusively to circulating CD169⁺ monocytes and Axl⁺ DCs. To determine the efficacy of ganglioside-liposomes for antigen presentation, we encapsulated peptides derived from the pancreatic cancer-associated tumor antigen Wilms tumor 1 (WT1) or melanoma-associated gp100 antigen into the ganglioside-liposomes. CD169⁺ moDCs and Axl⁺ DCs loaded with these ganglioside-liposomes efficiently activated CD8⁺ T cells specific for these epitopes. Moreover, Axl⁺ DCs were present in patients with four different cancers and could be targeted by ganglioside-liposomes. Our data demonstrate that ganglioside-liposomes can be used as nanovaccine carriers that efficiently target CD169⁺ DCs for cross-presentation and antigen-specific T cell activation. In conclusion, our studies support the concept that cancer vaccines targeting to CD169 can be applied to boost CD8⁺ T cell responses in cancer patients.     

Living with Advanced Chronic Obstructive Pulmonary Disease: A Qualitative Interview Study with Patients and Informal Carers

Abstract

The disease trajectory in chronic obstructive pulmonary disease (COPD) is characterised by a progressive decline in overall function, loss of independence and reduction of health-related quality of life. Although the symptom burden is high and care is often demanding, patients’ and informal carers’ experiences in living with advanced COPD are seldom described. This study sought to explore patients’ and informal carers’ experiences in living with advanced COPD and to understand their awareness about palliative care provision in advanced COPD. About 20 patients and 20 informal carers were recruited in a respiratory care service in Southern Switzerland. Semistructured individual interviews with participants were conducted on clinic premises and audio-recorded. Interviews lasted between 35 and 45?min. Data were analysed using thematic analysis.

Living day to day with COPD, psychosocial dimension of the disease and management of complex care were the main themes identified. Patients and informal carers reported a range of psychological challenges, with feelings of guilt, discrimination and blame. Most of the participants had no knowledge of palliative care and healthcare services did not provide them with any information about palliative care approaches in advanced COPD. The reported psychological challenges may influence the relationship between patients, informal carers and healthcare professionals, adding further complexity to the management of this long-term condition. Further research is needed to explore new ways of managing complex care in advanced COPD and to define how palliative care may be included in this complex care network.     

Proposal of an ultrasonographic classification for hepatic alveolar echinococcosis: Echinococcosis multilocularis Ulm classification-ultrasound

AIM: To establish an ultrasonographic classification based on a large sample of patients with confirmed hepatic alveolar echinococcosis(AE).METHODS: Clinical data and ultrasonography(US) findings of 185 patients(100 males; 85 females; mean age at diagnosis: 51.4 ± 17.6 years; mean age at time of US examination: 58.7 ± 18.2 years) were retrospectively reviewed with respect to the US morphology of hepatic AE lesions. The sonomorphological findings were grouped according to a five-part classification scheme.RESULTS: Application of the new classification resulted in the following distribution of sonomorphological patterns among the patients examined: hailstorm(54.1%); pseudocystic(13.5%); ossification(13.0%); hemangioma-like(8.1%); and metastasis-like(6.5%). Only 4.9% of lesions could not be assigned to a sonomorphological pattern.CONCLUSION: The sonomorphological classification proposed in the present study facilitates the diagnosis,interpretation and comparison of hepatic alveolar echinococcosis in routine practice and in the context of scientific studies.     

Cutaneous blood flow in type 2 diabetic individuals after an acute bout of maximal exercise

OBJECTIVE: We previously demonstrated a positive association between chronic aerobic exercise and dorsal foot skin blood flow during local heating in type 2 diabetic individuals. Thus, we hypothesized that a prior acute bout of maximal exercise would also have positive effects on postexercise blood flow. RESEARCH DESIGN AND METHODS: Subjects consisted of 32 individuals with type 2 diabetes and 26 nondiabetic control subjects further subdivided based on their physical activity status: diabetic exerciser (DE), diabetic sedentary (DS), control exerciser (CE), or control sedentary. Dorsal foot cutaneous blood flow was measured noninvasively by continuous laser-Doppler assessment at baseline and during local heating to 44 degrees C before and after a maximal bout of cycle exercise. Interstitial nitric oxide (NO) levels were measured concurrently in the foot dorsum. RESULTS: Increases in blood flow and its responsiveness to local heating to 44 degrees C were significantly lower in both diabetic groups compared with CE before maximal exercise, but perfusion responsiveness remained lower in DS subjects only after exercise (P < 0.05). Baseline skin blood flow was not different among groups preexercise, but it was significantly increased postexercise in DE subjects only. Interstitial NO levels were not significantly different at either time. At baseline, groups differed only in HbA(1c), fasting serum glucose, HDL cholesterol, and insulin resistance (homeostasis model assessment method). CONCLUSIONS: All diabetic individuals exhibit a blunted responsiveness of cutaneous blood flow with local heating to 44 degrees C before maximal exercise compared with active nondiabetic individuals, but after an exercise bout, it remains significantly blunted only in diabetic individuals who are sedentary. These findings occur independently of changes in interstitial NO levels.     

The dynamics of structural deformations of immobilized single light-harvesting complexes

Single assemblies of the intact light-harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.     

Disseminated neonatal herpes caused by herpes simplex virus types 1 and 2

Disseminated neonatal herpes simplex virus (HSV) infection is characterized by progressive multiple organ failure and high mortality rates. It can result from infection with either HSV-1 or HSV-2. We report a case of disseminated neonatal herpes that was caused by HSV-1 and HSV-2.