RESUMEN
Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.
Asunto(s)
Catepsina C , Coinfección , Granzimas , Infecciones por VIH , Macrófagos , Mycobacterium tuberculosis , Humanos , Granzimas/metabolismo , Granzimas/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/inmunología , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/virología , Coinfección/microbiología , Catepsina C/metabolismo , Catepsina C/genética , Cistatinas/metabolismo , Cistatinas/genética , Tuberculosis/metabolismo , Tuberculosis/inmunología , Tuberculosis/microbiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , VIH-1/fisiología , Biomarcadores de TumorRESUMEN
Breast cancer (BC) is the second-leading cause of cancer after lung cancer. The disease has affected millions of people and resulted in many deaths. In the metastasis of breast cancer cells, Topoisomerase IIα plays a vital role. Therefore, this investigation aims to identify potential flavonoid compounds against BC by inhibiting this enzyme at an early stage. Based on previous studies, we selected and screened several plant-derived flavonoid compounds with potential anti-breast cancer activity using PyRx 0.8 and Schrodinger applications for preliminary molecular docking: the highest docking scores of Myricetin (-11.6 kcal/mol) and Quercetin (-10.0 kcal/mol). Next, we evaluated the top four compounds on the Way2Drug server to complete the cytotoxicity evaluation, which demonstrated anti-cancer and anti-breast cancer activity in various cell lines. According to pharmacokinetics studies, four compounds exhibited outstanding values and functioned similar to drug-like molecules. Moreover, Myricetin, Quercetin, and Morin displayed the highest number of hydrogen bonds, with the corresponding receptor forming residues asn120, thr147, and lys168. The protein-ligand complexes were validated using the Desmond simulator, and their data were compared to the anti-breast cancer drug Doxorubicin. In the simulation analysis, various parameters were evaluated, including RMSD, RMSF, Rg, SASA, MolSA, PSA, and hydrogen bond interaction. Finally, validated our dynamic simulation result with MM-GBSA operation, and Myricetin and Quercetin had the greatest score of -72.74344651, -66.66771823 kcal/mol, which is outstanding than the control drug. Hence, the computational research approach determined that Myricetin, Quercetin, and Morin could be industrially developed for the alternative treatment of breast cancer following additional confirmation from animal and cell line studies.
RESUMEN
Tuberculosis (TB) treatment relies primarily on 70-year-old drugs, and prophylaxis suffers from the lack of an effective vaccine. Among the 10 million people exhibiting disease symptoms yearly, 450,000 have multidrug or extensively drug-resistant (MDR or XDR) TB. A greater understanding of host and pathogen interactions will lead to new therapeutic interventions for TB eradication. One of the strategies will be to target the host for better immune bactericidal responses against the TB causative agent Mycobacterium tuberculosis (Mtb). Cathepsins are promising targets due to their manipulation of Mtb with consequences such as decreased proteolytic activity and improved pathogen survival in macrophages. We recently demonstrated that we could overcome this enzymatic blockade by manipulating protease inhibitors such as cystatins. Here, we investigate the role of cystatin F, an inhibitor that we showed previously to be strongly upregulated during Mtb infection. Our results indicate that the silencing of cystatin F using siRNA increase the proteolytic activity of cathepsins S, L, and B, significantly impacting pathogen intracellular killing in macrophages. Taken together, these indicate the targeting of cystatin F as a potential adjuvant therapy for TB, including MDR and XDR-TB.
RESUMEN
Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the most successfully adapted human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arises. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pili, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle, Mtb induces necrotic cell death in macrophages to escape to the extracellular milieu and instructs a strong inflammatory response that is required for the progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This narrative review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish successful infection and transmission and its use as a target for the development of diagnostic tools and vaccines.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Tuberculosis/microbiologíaRESUMEN
Human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb) are pathogens responsible for millions of new infections each year; together, they cause high morbidity and mortality worldwide. In addition, late-stage HIV infection increases the risk of developing tuberculosis (TB) by a factor of 20 in latently infected people, and even patients with controlled HIV infection on antiretroviral therapy (ART) have a fourfold increased risk of developing TB. Conversely, Mtb infection exacerbates HIV pathogenesis and increases the rate of AIDS progression. In this review, we discuss this reciprocal amplification of HIV/Mtb coinfection and how they influence each other's pathogenesis. Elucidating the infectious cofactors that impact on pathogenesis may open doors for the design of new potential therapeutic strategies to control disease progression, especially in contexts where vaccines or the sterile clearance of pathogens are not effectively available.
RESUMEN
The golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.
RESUMEN
Ca2+, which provides binding sites for ligand water molecules W3 and W4 in the Mn4CaO5 cluster, is a prerequisite for O2 evolution in photosystem II (PSII). We report structural changes in the H-bond network and the catalytic cluster itself upon the replacement of Ca2+ with other alkaline earth metals, using a quantum mechanical/molecular mechanical approach. The small radius of Mg2+ makes W3 donate an H-bond to D1-Glu189 in Mg2+-PSII. If an additional water molecule binds at the large surface of Ba2+, it donates H-bonds to D1-Glu189 and the ligand water molecule at the dangling Mn, altering the H-bond network. The potential energy profiles of the H-bond between D1-Tyr161 (TyrZ) and D1-His190 and the interconversion between the open- and closed-cubane S2 conformations remain substantially unaltered upon the replacement of Ca2+. Remarkably, the O5â¯Ca2+ distance is shortest among all O5â¯metal distances irrespective of the radius being larger than that of Mg2+. Furthermore, Ca2+ is the only alkaline earth metal that equalizes the O5â¯metal and O2â¯metal distances and facilitates the formation of the symmetric cubane structure.
RESUMEN
Mycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Catepsinas/metabolismo , Saquinavir/farmacología , Saquinavir/metabolismo , Liposomas/metabolismo , Macrófagos/metabolismo , Tuberculosis/microbiología , Interacciones Huésped-Patógeno/fisiologíaRESUMEN
Cathepsins were first described, as endolysosomal proteolytic enzymes in reference to the organelles where they degrade the bulk of endogenous and exogenous substrates in a slightly acidic environment. These substrates include pathogens internalized via endocytosis and/or marked for destruction by autophagy. However, the role of cathepsins during infection far exceeds that of direct digestion of the pathogen. Cathepsins have been extensively investigated in the context of tumour associated immune cells and chronic inflammation. Several cathepsin-dependent immune responses develop in the endocytic pathway while others take place in the cytosol, the nucleus, or in the extracellular space. In this review we highlight the spatial localization of cathepsins and their implications in immune activation and resolution pathways during infection.
Asunto(s)
Catepsinas , Lisosomas , Catepsinas/metabolismo , Endocitosis , Endosomas/metabolismo , Humanos , Inflamación/metabolismo , Lisosomas/metabolismoRESUMEN
India is affected strongly by the Coronavirus and within a short period, it becomes the second-highest country based on the infected case. Earlier, there was an indication of the impact of pollution on COVID-19 transmission from a few studies with early COVID-19 data. The study of the effect of pollution on COVID-19 in Indian metropolitan cities is ideal due to the high level of pollution and COVID-19 transmission in these cities. We study the impact of different air pollutants on the spread of coronavirus in different cities in India. A correlation is studied with daily confirmed COVID-19 cases with a daily mean of ozone, particle matter (PM) in size ≤ 10 µ m, carbon monoxide, sulfur dioxide, and nitrogen dioxide of different cities. It is found that particulate matter concentration decreases during the nationwide lockdown period and the air quality index improves for different Indian regions. A correlation between the daily confirmed cases with particulate matter (PM 2.5 and PM 10 both) is observed. The air quality index also shows a positive correlation with the daily confirmed cases for most of the metropolitan Indian cities. The correlation study also indicates that different air pollutants may have a role in the spread of the virus.
RESUMEN
Coronavirus Disease-2019 (COVID-19) started in Wuhan province of China in November 2019 and within a short time, it was declared as a worldwide pandemic by World Health Organisation due to the very fast worldwide spread of the virus. There are a few studies that look for the correlation with infected individuals and different environmental parameters using early data of COVID-19 but there is no study so far that deals with the variation of effective reproduction number and environmental factors. Effective reproduction number is the driving parameter of the spread of a pandemic and it is important to study the effect of various environmental factors on effective reproduction number to understand the effect of those factors on the spread of the virus. We have used time-dependent models to investigate the variation of different time-dependent driving parameters of COVID-19 like effective reproduction number and contact rate using data from India as a test case. India is a large population country that is highly affected due to the COVID-19 pandemic and has a wide span of different temperature and humidity regions and is ideal for such study. We have studied the impact of temperature and humidity on the spread of the virus of different Indian states using time-dependent epidemiological models SIRD, and SEIRD for a long time scale. We have used a linear regression method to look for any dependency between the effective reproduction number with the relative humidity, absolute humidity, and temperature. The effective reproduction number shows a negative correlation with both relative and absolute humidity for most of the Indian states, which are statistically significant. This implies that relative and absolute humidity may have an important role in the variation of effective reproduction number. Most of the states (six out of ten) show a positive correlation while two (out of ten) show a negative correlation between effective reproduction number and average air temperature for both SIRD and SEIRD models.
Asunto(s)
COVID-19 , Número Básico de Reproducción , COVID-19/epidemiología , Humanos , Humedad , Pandemias , SARS-CoV-2 , TemperaturaRESUMEN
Pulmonary fibrosis (PF) is a disease-refractive lung condition with an increased rate of mortality. The potential factors causing PF include viral infections, radiation exposure, and toxic airborne chemicals. Idiopathic PF (IPF) is related to pneumonia affecting the elderly and is characterized by recurring scar formation in the lungs. An impaired wound healing process, defined by the dysregulated aggregation of extracellular matrix components, triggers fibrotic scar formation in the lungs. The potential pathogenesis includes oxidative stress, altered cell signaling, inflammation, etc. Nintedanib and pirfenidone have been approved with a conditional endorsement for the management of IPF. In addition, natural product-based treatment strategies have shown promising results in treating PF. In this study, we reviewed the recently published literature and discussed the potential uses of natural products, classified into three types-isolated active compounds, crude extracts of plants, and traditional medicine, consisting of mixtures of different plant products-in treating PF. These natural products are promising in the treatment of PF via inhibiting inflammation, oxidative stress, and endothelial mesenchymal transition, as well as affecting TGF-ß-mediated cell signaling, etc. Based on the current review, we have revealed the signaling mechanisms of PF pathogenesis and the potential opportunities offered by natural product-based medicine in treating PF.
Asunto(s)
Productos BiológicosRESUMEN
The production of biopharmaceuticals in engineered plant-based systems is a promising technology that has proven its suitability for the production of various recombinant glyco-proteins that are currently undergoing clinical trials. However, compared to mammalian cell lines, the productivity of plant-based systems still requires further improvement. A major obstacle is the proteolytic degradation of recombinant target proteins by endogenous plant proteases mainly from the subtilisin family of serine proteases. In the present study, the authors screened for putative small molecule inhibitors for subtilases that are secreted from tobacco BY-2 suspension cells using an in silico approach. The effectiveness of the substances identified in this screen was subsequently tested in degradation assays using the human broadly-neutralizing anti-HIV monoclonal antibody 2F5 (mAb2F5) and spent BY-2 culture medium as a model system. Among 16 putative inhibitors identified by in silico studies, three naphthalene sulfonic acid derivatives showed inhibitory activity in in vitro degradation assays and are similar to or even more effective than phenylmethylsulfonyl fluoride (PMSF), a classical inhibitor of serine proteases, which served as positive control.
Asunto(s)
Anticuerpos Anti-VIH , VIH-1 , Animales , Anticuerpos Anti-VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Mamíferos , Proteínas Recombinantes/metabolismo , Serina Proteasas/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
In photosystem II (PSII), Cl- is a prerequisite for the second flash-induced oxidation of the Mn4CaO5 cluster (the S2 to S3 transition). We report proton transfer from the substrate water molecule via D1-Asp61 and electron transfer via redox-active D1-Tyr161 (TyrZ) to the chlorophyll pair in Cl--depleted PSII using a quantum mechanical/molecular mechanical approach. The low-barrier H-bond formation between the substrate water molecule and D1-Asp61 remained unaffected upon the depletion of Cl-. However, the binding site, D2-Lys317, formed a salt bridge with D1-Asp61, leading to the inhibition of the subsequent proton transfer. Remarkably, the redox potential (Em) of S2/S3 increased significantly, making electron transfer from S2 to TyrZ energetically uphill, as observed in Ca2+-depleted PSII. The uphill electron transfer pathway was induced by the significant increase in Em(S2/S3) caused by the loss of charge compensation for D2-Lys317 upon the depletion of Cl-, whereas it was induced by the significant decrease in Em(TyrZ) caused by the rearrangement of the water molecules at the Ca2+ binding moiety upon the depletion of Ca2+.
Asunto(s)
Cloruros , Agua , Electrones , Oxidación-Reducción , Oxígeno , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
India is one of the countries in the world which is badly affected by the COVID-19 second wave. Assembly election in four states and a union territory of India was taken place during March-May 2021 when the COVID-19 second wave was close to its peak and affected a huge number of people. We studied the impact of assembly election on the effective contact rate and the effective reproduction number of COVID-19 using different epidemiological models like SIR, SIRD, and SEIR. We also modeled the effective reproduction number for all election-bound states using different mathematical functions. We separately studied the case of all election-bound states and found all the states showed a distinct increase in the effective contact rate and the effective reproduction number during the election-bound time and just after that compared to pre-election time. States, where elections were conducted in single-phase, showed less increase in the effective contact rate and the reproduction number. The election commission imposed extra measures from the first week of April 2021 to restrict big campaign rallies, meetings, and different political activities. The effective contact rate and the reproduction number showed a trend to decrease for few states due to the imposition of the restrictions. We also compared the effective contact rate, and the effective reproduction number of all election-bound states and the rest of India and found all the parameters related to the spread of virus for election-bound states are distinctly high compared to the rest of India.
RESUMEN
In photosystem II (PSII), the second-lowest oxidation state (S1) of the oxygen-evolving Mn4CaO5 cluster is the most stable, as the radical form of the redox-active D2-Tyr160 is considered to be a candidate that accepts an electron from the lowest oxidation state (S0) in the dark. Using quantum mechanical/molecular mechanical calculations, we investigated the redox potential (E m) of TyrD and its H-bond partner, D2-His189. The potential energy profile indicates that the release of a proton from the TyrD...D2-His189 pair leads to the formation of a low-barrier H-bond. The E m depends on the H+ position along the low-barrier H-bond, e.g., 680 mV when the H+ is at the D2-His189 moiety and 800 mV when the H+ is at the TyrD moiety, which can explain why TyrD mediates both the S0 to S1 oxidation and the S2 to S1 reduction.
RESUMEN
Tuberculosis owes its resurgence as a major global health threat mostly to the emergence of drug resistance and coinfection with HIV. The synergy between HIV and Mycobacterium tuberculosis (Mtb) modifies the host immune environment to enhance both viral and bacterial replication and spread. In the lung immune context, both pathogens infect macrophages, establishing favorable intracellular niches. Both manipulate the endocytic pathway in order to avoid destruction. Relevant players of the endocytic pathway to control pathogens include endolysosomal proteases, cathepsins, and their natural inhibitors, cystatins. Here, a mapping of the human macrophage transcriptome for type I and II cystatins during Mtb, HIV, or Mtb-HIV infection displayed different profiles of gene expression, revealing cystatin C as a potential target to control mycobacterial infection as well as HIV coinfection. We found that cystatin C silencing in macrophages significantly improves the intracellular killing of Mtb, which was concomitant with an increased general proteolytic activity of cathepsins. In addition, downmodulation of cystatin C led to an improved expression of the human leukocyte antigen (HLA) class II in macrophages and an increased CD4+ T-lymphocyte proliferation along with enhanced IFN-γ secretion. Overall, our results suggest that the targeting of cystatin C in human macrophages represents a promising approach to improve the control of mycobacterial infections including multidrug-resistant (MDR) TB.
Asunto(s)
Coinfección/inmunología , Cistatina C/inmunología , Infecciones por VIH/inmunología , Macrófagos/inmunología , Tuberculosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Cistatina C/genética , VIH-1 , Humanos , Interferón gamma/inmunología , Macrófagos/microbiología , Mycobacterium tuberculosisRESUMEN
Photosystem II allows water to be the primary electron source for the photosynthetic electron transfer chain. Water is oxidized to dioxygen at the Oxygen Evolving Complex (OEC), a Mn4CaO5 inorganic core embedded on the lumenal side of PSII. Water-filled channels surrounding the OEC must bring in substrate water molecules, remove the product protons to the lumen, and may transport the product oxygen. Three water-filled channels, denoted large, narrow, and broad, extend from the OEC towards the aqueous surface more than 15 Å away. However, the role of each pathway in the transport in and out of the OEC is yet to be established. Here, we combine Molecular Dynamics (MD), Multi Conformation Continuum Electrostatics (MCCE) and Network Analysis to compare and contrast the three potential proton transfer paths. Hydrogen bond network analysis shows that near the OEC the waters are highly interconnected with similar free energy for hydronium at all locations. The paths diverge as they move towards the lumen. The water chain in the broad channel is better connected than in the narrow and large channels, where disruptions in the network are observed approximately 10 Å from the OEC. In addition, the barrier for hydronium translocation is lower in the broad channel. Thus, a proton released from any location on the OEC can access all paths, but the likely exit to the lumen passes through PsbO via the broad channel.
Asunto(s)
Electrones , Oxígeno/química , Fotosíntesis , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Agua/química , Enlace de Hidrógeno , Oxidación-Reducción , Oxígeno/metabolismo , Agua/metabolismoRESUMEN
Photosystem II (PSII) contains Ca2+, which is essential to the oxygen-evolving activity of the catalytic Mn4CaO5 complex. Replacement of Ca2+ with other redox-inactive metals results in a loss/decrease of oxygen-evolving activity. To investigate the role of Ca2+ in this catalytic reaction, we investigate artificial Mn3[M]O2 clusters redox-inactive metals [M] ([M] = Mg2+, Ca2+, Zn2+, Sr2+, and Y3+), which were synthesized by Tsui et al. (Nat Chem 5:293, 2013). The experimentally measured redox potentials (Em) of these clusters are best described by the energy of their highest occupied molecular orbitals. Quantum chemical calculations showed that the valence of metals predominantly affects Em(MnIII/IV), whereas the ionic radius of metals affects Em(MnIII/IV) only slightly.
Asunto(s)
Calcio/química , Transporte de Electrón , Compuestos de Manganeso/química , Manganeso/química , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/químicaRESUMEN
We report the existence of two distinct oxygen-radical-containing Mn4CaO5/6 conformations with short O···O bonds in the crystal structures of the oxygen-evolving enzyme photosystem II (PSII), obtained using an X-ray free electron laser (XFEL). A short O···O distance of <2.3 Å between the O4 site of the Mn4CaO5 complex and the adjacent water molecule (W539) in the proton-conducting O4-water chain was observed in the second flash-induced (2F) XFEL structure (2F-XFEL), which may correspond to S3. By use of a quantum mechanical/molecular mechanical approach, the OH⢠formation at W539 and the short O4···OW539 distance (<2.3 Å) were reproduced in S2 and S3 with reduced Mn1(III), which lacks the additional sixth water molecule O6. As the Oâ¢- formation at O6 and the short O5···O6 distance (1.9 Å) have been reported in another 2F-XFEL structure with reduced Mn4(III), two distinct oxygen-radical conformations exist in the 2F-XFEL crystals.