Thermally induced gluten modification observed with rheology and spectroscopies

The protein vital gluten is mainly used for food while interest for non-food applications, like biodegradable materials, increases. In general, the structure and functionality of proteins is highly dependent on thermal treatments during production or modification. This study presents conformational changes and corresponding rheological effects of vital wheat gluten depending on temperature. Dry samples analyzed by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR) and thermalgravimetric analysis coupled with mass spectrometry (TGA-MS) show surface compositions and conformational changes from 25 to 250 °C. Above 170 °C, XPS reveals a decreased N content at the surface while FTIR band characteristics for β-sheets prove structural changes. At 250 °C, protein denaturation accompanied by a significant mass loss due to dehydration and decarbonylation reactions is observed. Oscillatory measurements of optimally hydrated vital gluten describing network properties of the material show two structural changes along a temperature ramp from 25 to 90 °C: at 56–64 °C, the temperature necessary to trigger structural changes increases with the ratio of gliadin to total protein mass, determined by reversed-phase high performance liquid chromatography (RP-HPLC). At a temperature of 79–81 °C, complete protein denaturation occurs. FTIR confirms the denaturation process by showing band shifts with both temperature steps

Continuous monitoring of bread dough fermentation using a 3D vision Structured Light technique

Fermentation of the dough is an important phase in the bread-making process which is affected by several important factors related to raw materials and processing. Changes in fermentation affect parameters in the final product, such as texture, palatability and general quality. For this reason, it is important to develop dynamic methods to study this phase. In this work, a 3D vision system based on Structured Light (SL) was used to monitor the fermentation phase. The evolution of the dough was studied employing 10 wheat flours with non-physicochemical and rheological differences. However, differences in dough behaviors during fermentation were found based on SL method parameters. When the variation of the total transversal area was related to the maximum height at each fermentation time a set of peaks and valleys appeared. These sets were directly related to the fermentation capacity. Specifically, a lower number of peaks during the main fermentation time (100 min) is related to wheat flours with high fermentation capacity. Consequently, the proposed SL Technique could be used as a method to check the fermentation capacity of wheat flours according to their fermentation behavior.We thank the Polytechnic University of Valencia and Generalitat Valenciana for the financial support provided (PAID-05-011-2870 and 226 GVPRE/2008/170 projects, respectively)Ivorra Martínez, E.; Verdú Amat, S.; Sánchez Salmerón, AJ.; Barat Baviera, JM.; Grau Meló, R. (2014). Continuous monitoring of bread dough fermentation using a 3D vision Structured Light technique. Journal of Food Engineering. 130:8-13. https://doi.org/10.1016/j.jfoodeng.2013.12.031S81313

The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir.

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC50) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors

Coreceptor Usage by HIV-1 and HIV-2 Primary Isolates: The Relevance of CCR8 Chemokine Receptor as an Alternative Coreceptor

The human immunodeficiency virus replication cycle begins by sequential interactions between viral envelope glycoproteins with CD4 molecule and a member of the seven-transmembrane, G-protein-coupled, receptors' family (coreceptor). In this report we focused on the contribution of CCR8 as alternative coreceptor for HIV-1 and HIV-2 isolates. We found that this coreceptor was efficiently used not only by HIV-2 but particularly by HIV-1 isolates. We demonstrate that CXCR4 usage, either alone or together with CCR5 and/or CCR8, was more frequently observed in HIV-1 than in HIV-2 isolates. Directly related to this is the finding that the non-usage of CXCR4 is significantly more common in HIV-2 isolates; both features could be associated with the slower disease progression generally observed in HIV-2 infected patients. The ability of some viral isolates to use alternative coreceptors besides CCR5 and CXCR4 could further impact on the efficacy of entry inhibitor therapy and possibly also in HIV pathogenesis

Contrasting Roles for TLR Ligands in HIV-1 Pathogenesis

The first line of a host's response to various pathogens is triggered by their engagement of cellular pattern recognition receptors (PRRs). Binding of microbial ligands to these receptors leads to the induction of a variety of cellular factors that alter intracellular and extracellular environment and interfere directly or indirectly with the life cycle of the triggering pathogen. Such changes may also affect any coinfecting microbe. Using ligands to Toll-like receptors (TLRs) 5 and 9, we examined their effect on human immunodeficiency virus (HIV)-1 replication in lymphoid tissue ex vivo. We found marked differences in the outcomes of such treatment. While flagellin (TLR5 agonist) treatment enhanced replication of CC chemokine receptor 5 (CCR 5)-tropic and CXC chemokine receptor 4 (CXCR4)-tropic HIV-1, treatment with oligodeoxynucleotide (ODN) M362 (TLR9 agonist) suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1-infected tissues. The nature and/or magnitude of these changes were dependent on the ligand as well as on the HIV-1 viral strain. Moreover, the tested ligands differed in their ability to induce cellular activation as evaluated by the expression of the cluster of differentiation markers (CD) 25, CD38, CD39, CD69, CD154, and human leukocyte antigen D related (HLA)-DR as well as of a cell proliferation marker, Ki67, and of CCR5. No significant effect of the ligand treatment was observed on apoptosis and cell death/loss in the treated lymphoid tissue ex vivo. Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention

A novel potassium channel in lymphocyte mitochondria

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance

Efficacy of NVC-422 against Staphylococcus aureus biofilms in a sheep biofilm model of sinusitis

BackgroundBacterial biofilms are a major obstacle in management of recalcitrant chronic rhinosinusitis. NVC-422 is a potent, fast-acting, broad-spectrum, nonantibiotic, antimicrobial with a new mechanism of action effective against biofilm bacteria in in vitro conditions. The aim of this study was to investigate the safety and efficacy of NVC-422 as local antibiofilm treatment in a sheep model of rhinosinusitis.MethodsAfter accessing and occluding frontal sinus ostia in 24 merino sheep via staged endoscopic procedures, S. aureus clinical isolate was instilled in frontal sinuses. Following biofilm formation, ostial obstruction was removed and sinuses irrigated with 0.1% and 0.5% NVC-422 in 5 mM acetate isotonic saline at pH 4.0. Sheep were monitored for adverse effects and euthanized 24 hours after treatment. Frontal sinuses were assessed for infection and changes in mucosa after the treatment. S. aureus biofilms were identified with Baclight-confocal scanning microscopy protocol and the biofilm biomass assayed by applying the COMSTAT2 program to recorded image stacks.ResultsAfter 2 irrigations with 0.1% NVC-422, S. aureus biofilm biomass was reduced when compared to control sinuses (p = 0.0001), though this effect was variable in samples. NVC-422 0.5% solution irrigations reduced biofilm even more significantly and consistently over all samples (p ConclusionNVC-422 is an effective topical agent against S. aureus biofilms, with dose-dependent efficacy in this animal model of biofilm-associated sinusitis.Deepti Singhal, Andreas Jekle, Dmitri Debabov, Lu Wang, Bez Khosrovi, Mark Anderson, Andrew Foreman, and Peter-John Wormal

Coreceptor Phenotype of Natural Human Immunodeficiency Virus with Nef Deleted Evolves In Vivo, Leading to Increased Virulence

The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the nef long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4(+) T cells than a reference dualtropic, nef-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4(+) T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5(+) CD4(+) T cells, the cytotoxic effect of the 1999 isolate toward CCR5(−) CD4(+) T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5(+) and CCR5(−) CXCR4(+) cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5(+) cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this nef-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS