Evaluating the epizootic and zoonotic threat of an H7N9 low-pathogenicity avian influenza virus (LPAIV) variant associated with enhanced pathogenicity in turkeys.

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses.

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

Building bridges between fields: bringing together development and homeostasis.

Despite striking parallels between the fields of developmental biology and adult tissue homeostasis, these are disconnected in contemporary research. Although development describes tissue generation and homeostasis describes tissue maintenance, it is the balance between stem cell proliferation and differentiation that coordinates both processes. Upstream signalling regulates this balance to achieve the required outcome at the population level. Both development and homeostasis require tight regulation of stem cells at the single-cell level and establishment of patterns at the tissue-wide level. Here, we emphasize that the general principles of embryonic development and tissue homeostasis are similar, and argue that interactions between these disciplines will be beneficial for both research fields.

A New Look at the Mechanism of Cocrystal Formation and Coformers Exchange in Processes Forced by Mechanical and/or Thermal Stimuli - ex situ and in†situ Studies of Low-Melting Eutectic Mixtures.

Three different devices: ball mill, hot stage melting, and magic angle spinning (MAS) NMR rotor were used for the preparation of ethenzamide (ET) cocrystals with glutaric acid (GLU), ethylmalonic acid (EMA) and maleic acid (MAL) as coformers. In each case, well-defined binary systems (ET:EMA, ET:GLU, ET:MAL) were obtained. The common features of the two solvent free methods of cocrystal formation (grinding, melting) are presented on the basis of arguments obtained by solid state NMR spectroscopy. Thermal analysis (Differential Scanning Calorimetry) proved that the eutectic phase arises over a wide range of molar ratios of components for each of the binary systems. NMR techniques, supported by theoretical calculations, allowed to provide details about the pathway of the reaction mechanism with atomic accuracy. It was found that the formation of ET cocrystals is a complex process that requires five steps. Each step has been recognized and described. Variable temperature 1D and 2D MAS NMR experiments allowed to track physicochemical processes taking place in a molten state. Moreover, it was found that in a multicomponent mixture consisting of all four components, ET, EMA, GLU, and MAL, ET in the molten phase behaves as a specific selector choosing only one partner to form binary cocrystals according to energy preferences. The process of exchange of coformers in binary systems during grinding, melting, and NMR measurements is described. The stabilization energies (Estab ) and molecular electrostatic potential (MEP) maps computed for the cocrystals under discussion and their individual components rationalize the selection rules and explain the relationships between individual species.

Solvent-Free Mechanosynthesis of Oligopeptides by Coupling Peptide Segments of Different Lengths - Elucidating the Role of Cesium Carbonate in Ball Mill Processes.

We report an idea for the synthesis of oligopeptides using a solvent-free ball milling approach. Our concept is inspired by block play, in which it is possible to construct different objects using segments (blocks) of different sizes and lengths. We prove that by having a library of short peptides and employing the ball mill mechanosynthesis (BMMS) method, peptides can be easily coupled to form different oligopeptides with the desired functional and biological properties. Optimizing the BMMS process we found that the best yields we obtained when TBTU and cesium carbonate were used as reagents. The role of Cs2CO3 in the coupling mechanism was followed on each stage of synthesis by 1H, 13C and 133Cs NMR employing Magic Angle Spinning (MAS) techniques. It was found that cesium carbonate acts not only as a base but is also responsible for the activation of substrates and intermediates. The unique information about the BMMS mechanism is based on the analysis of 2D NMR data. The power of BMMS is proved by the example of different peptide combinations, 2+2, 3+2, 4+2, 5+2 and 4+4. The tetra-, penta-, hexa-, hepta- and octapeptides obtained under this project were fully characterized by MS and NMR techniques.

Mixed-phase weak anion-exchange/reversed-phase LC-MS/MS for analysis of nucleotide sugars in human fibroblasts.

Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-ß-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond.

Clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) from the 2021/22 epizootic is highly duck adapted and poorly adapted to chickens.

The 2021/2022 epizootic of high pathogenicity avian influenza (HPAIV) remains one of the largest ever in the UK, being caused by a clade 2.3.4.4b H5N1 HPAIV. This epizootic affected more than 145 poultry premises, most likely through independent incursion from infected wild birds, supported by more than 1700 individual detections of H5N1 from wild bird mortalities. Here an H5N1 HPAIV, representative of this epizootic (H5N1-21), was used to investigate its virulence, pathogenesis and transmission in layer chickens and Pekin ducks, two species of epidemiological importance. We inoculated both avian species with decreasing H5N1-21 doses. The virus was highly infectious in ducks, with high infection levels and accompanying shedding of viral RNA, even in ducks inoculated with the lowest dose, reflecting the strong waterfowl adaptation of the clade 2.3.4.4 HPAIVs. Duck-to-duck transmission was very efficient, coupled with high environmental contamination. H5N1-21 was frequently detected in water sources, serving as likely sources of infection for ducks, but inhalable dust and aerosols represented low transmission risks. In contrast, chickens inoculated with the highest dose exhibited lower rates of infection compared to ducks. There was no evidence for experimental H5N1-21 transmission to any naive chickens, in two stocking density scenarios, coupled with minimal and infrequent contamination being detected in the chicken environment. Systemic viral dissemination to multiple organs reflected the pathogenesis and high mortalities in both species. In summary, the H5N1-21 virus is highly infectious and transmissible in anseriformes, yet comparatively poorly adapted to galliformes, supporting strong host preferences for wild waterfowl. Key environmental matrices were also identified as being important in the epidemiological spread of this virus during the continuing epizootic.

The Origin of Internal Genes Contributes to the Replication and Transmission Fitness of H7N9 Avian Influenza Virus.

H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate A/Anhui/1/13 (H7N9) and three reassortant viruses (2:6 H7N9) which contained the hemagglutinin and neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs belonging to (i) G1 subgroup 2, (ii) G1 subgroup 3, or (iii) BJ94 lineages, enzootic in different regions throughout Asia. Infection of chickens with the 2:6 H7N9 containing G1-like H9N2 internal genes conferred attenuation in vivo, with reduced shedding and transmission to contact chickens. However, possession of BJ94-like H9N2 internal genes resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis showed that the 2:6 H7N9 with BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo coinfection experiments followed, where chickens were coinfected with pairs of Anhui/13 H7N9 and a 2:6 H7N9 reassortant. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 AIVs with internal gene segments of BJ94-like or G1-like H9N2 viruses. Coinfection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens, and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential. IMPORTANCE H9N2 avian influenza viruses (AIVs) are enzootic in poultry in different geographical regions. The internal genes of these viruses can be exchanged with other zoonotic AIVs, most notably the A/Anhui/1/2013-lineage H7N9, which can give rise to new virus genotypes with increased veterinary, economic and public health threats to both poultry and humans. We investigated the propensity of the internal genes of H9N2 viruses (G1 or BJ94) in the generation of novel reassortant H7N9 AIVs. We observed that the internal genes of H7N9 which were derivative of BJ94-like H9N2 virus have a fitness advantage compared to those from the G1-like H9N2 viruses for efficient transmission among chickens. We also observed the generation of novel reassortant viruses during chicken transmission which infected and replicated efficiently in human cells. Therefore, such emergent reassortant genotypes may pose an elevated zoonotic threat.

Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential.

An H7N9 low-pathogenicity avian influenza virus (LPAIV) emerged in 2013 through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. To examine a natural reassortment scenario between H7N9 and G1 lineage H9N2 viruses predominant in the Indian subcontinent, we performed an experimental coinfection of chickens with A/Anhui/1/2013/H7N9 (Anhui/13) virus and A/Chicken/Pakistan/UDL-01/2008/H9N2 (UDL/08) virus. Plaque purification and genotyping of the reassortant viruses shed via the oropharynx of contact chickens showed H9N2 and H9N9 as predominant subtypes. The reassortant viruses shed by contact chickens also showed selective enrichment of polymerase genes from H9N2 virus. The viable "6+2" reassortant H9N9 (having nucleoprotein [NP] and neuraminidase [NA] from H7N9 and the remaining genes from H9N2) was successfully shed from the oropharynx of contact chickens, plus it showed an increased replication rate in human A549 cells and a significantly higher receptor binding to α2,6 and α2,3 sialoglycans compared to H9N2. The reassortant H9N9 virus also had a lower fusion pH, replicated in directly infected ferrets at similar levels compared to H7N9 and transmitted via direct contact. Ferrets exposed to H9N9 via aerosol contact were also found to be seropositive, compared to H7N9 aerosol contact ferrets. To the best of our knowledge, this is the first study demonstrating that cocirculation of H7N9 and G1 lineage H9N2 viruses could represent a threat for the generation of novel reassortant H9N9 viruses with greater virulence in poultry and a zoonotic potential. IMPORTANCE We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of the G1 lineage that are enzootic in poultry across the Indian subcontinent and the Middle East. Coinfection of chickens with these viruses resulted in the emergence of novel reassortant H9N9 viruses with genes derived from both H9N2 and H7N9 viruses. The "6+2" reassortant H9N9 (having NP and NA from H7N9) virus was shed from contact chickens in a significantly higher proportion compared to most of the reassortant viruses, showed significantly increased replication fitness in human A549 cells, receptor binding toward human (α2,6) and avian (α2,3) sialic acid receptor analogues, and the potential to transmit via contact among ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with zoonotic potential.

Unexpected Factors Affecting the Kinetics of Guest Molecule Release from Investigation of Binary Chemical Systems Trapped in a Single Void of Mesoporous Silica Particles.

In this work, we present results for loading of well-defined binary systems (cocrystal, solid solution) and untreated materials (physical mixtures) into the voids of MCM-41 mesoporous silica particles employing three different filling methods. The applied techniques belong to the group of "wet methods" (diffusion supported loading - DiSupLo) and "solvent-free methods" (mechanical ball-mill loading - MeLo, thermal solvent free - TSF). As probes for testing the guest1-guest2 interactions inside the MCM-41 pores we employed the benzoic acid (BA), perfluorobenzoic acid (PFBA), and 4-fluorobenzoic acid (4-FBA). The guests intermolecular contacts and phase changes were monitored employing magic angle spinning (MAS) NMR Spectroscopy techniques and powder X-ray diffraction (PXRD). Since mesoporous silica materials are commonly used in drug delivery system research, special attention has been paid to factors affecting guest release kinetics. It has been proven that not only the content and composition of binary systems, but also the loading technique have a strong impact on the rate of guests release. Innovative methods of visualizing differences in release kinetics are presented.

Double aortic arch: implications of antenatal diagnosis, differential growth of arches during pregnancy, associated abnormalities and postnatal outcome.

OBJECTIVES: To evaluate the prenatal characteristics of double aortic arch (DAA), assess the relative size of the arches and their growth during pregnancy, describe associated cardiac, extracardiac and chromosomal/genetic abnormalities and review postnatal presentation and clinical outcome. METHODS: This was a retrospective cohort study of all fetuses with a confirmed diagnosis of DAA seen in five specialized referral centers in London, UK, between October 2012 and November 2019. Cases were identified from the hospitals' fetal databases. Fetal echocardiographic findings, intracardiac and extracardiac abnormalities, genetic defects, computed tomography (CT) findings and postnatal clinical presentation and outcome were evaluated. RESULTS: A total of 79 fetuses with DAA were included. Of those assessed postnatally, 48.6% had an atretic left aortic arch (LAA), while 5.1% had an atretic LAA at the first fetal scan and were misdiagnosed antenatally with right aortic arch (RAA). The LAA was atretic in 55.8% of those who underwent CT. DAA was an isolated abnormality in 91.1% of cases; 8.9% of patients had an additional intracardiac abnormality and 2.5% had both intra- and extracardiac abnormalities. Among the 52 cases that underwent genetic testing, 11.5% had genetic abnormalities and, specifically, the 22q11 microdeletion was identified in 3.8% of patients. At a median follow-up of 993.5 days, 42.5% of patients had developed symptoms of tracheoesophageal compression (5.5% during the first month after birth) and 56.2% had undergone intervention. Statistical analysis using the χ-square test showed no significant relationship between morphology of DAA (patency of both aortic arches vs atretic LAA) and the need for intervention (P = 0.134), development of vascular ring symptoms (P = 0.350) or evidence of airway compression on CT (P = 0.193). CONCLUSIONS: Most cases of DAA can be diagnosed easily at midgestation, as typically both arches are patent with a dominant RAA at this stage. However, we found that the LAA had become atretic in approximately half of the cases postnatally, supporting the theory of differential growth of the arches during pregnancy. DAA is usually an isolated abnormality; however, thorough assessment is required to exclude associated intra- and extracardiac anomalies and to determine the need for invasive prenatal genetic testing. Postnatally, early clinical assessment is needed and CT scan should be considered, irrespective of the presence of symptoms. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

The Clinical Application of Immunohistochemical Expression of Notch4 Protein in Patients with Colon Adenocarcinoma.

The Notch signalling pathway is one of the most conserved and well-characterised pathways involved in cell fate decisions and the development of many diseases, including cancer. Among them, it is worth noting the Notch4 receptor and its clinical application, which may have prognostic value in patients with colon adenocarcinoma. The study was performed on 129 colon adenocarcinomas. Immunohistochemical and fluorescence expression of Notch4 was performed using the Notch4 antibody. The associations between the IHC expression of Notch4 and clinical parameters were analysed using the Chi2 test or Chi2Yatesa test. The Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Notch4 expression and the 5-year survival rate of patients. Intracellular localisation of Notch4 was detected by the use of the immunogold labelling method and TEM. 101 (78.29%) samples had strong Notch4 protein expression, and 28 (21.71%) samples were characterised by low expression. The high expression of Notch4 was clearly correlated with the histological grade of the tumour (p < 0.001), PCNA immunohistochemical expression (p < 0.001), depth of invasion (p < 0.001) and angioinvasion (p < 0.001). We can conclude that high expression of Notch4 is correlated with poor prognosis of colon adenocarcinoma patients (log-rank, p < 0.001).

Synthesis and Characterization of Antibacterial Chitosan Films with Ciprofloxacin in Acidic Conditions.

This work presents the results of research on obtaining chitosan (CS) films containing on their surface ciprofloxacin (CIP). A unique structure was obtained that not only gives new properties to the films, but also changes the way of coverage and structure of the surface. The spectroscopic test showed that in the process of application of CIP on the surface of CS film, CIP was converted from its crystalline form to an amorphic one, hence improving its bioavailability. This improved its scope of microbiological effect. The research was carried out on the reduction of CIP concentration during the process of CIP adhesion to the surface of chitosan films. The antibacterial activity of the CS films with and without the drug was evaluated in relation to Escherichia coli and Staphylococcus aureus, as well as Candida albicans and Penicillium expansum. Changes in the morphology and roughness of membrane surfaces after the antibacterial molecule adhesion process were tested with atomic force microscopy (AFM) and scanning electron microscopy (SEM). Structural analysis of CS and its modifications were confirmed with Fourier-transform spectroscopy in the infrared by an attenuated total reflectance of IR radiation (FTIR-ATR) and solid-state nuclear magnetic resonance (NMR).

In Vitro Skeletal Muscle Model of PGM1 Deficiency Reveals Altered Energy Homeostasis.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

The growing role of telemedicine - possibilities and regulations concerning teleradiology in Poland.

In recent years, imaging studies have become increasingly used at various stages in the management of patients with various conditions and disorders. This process results in a necessity to provide an increasing number of exams, which involves a growing role of radiologists in assessing and reporting those exams. The article discusses tele-radiology as a method that can improve access to radiology services, presenting its potential benefits, as well as the risks involved. It analyses access to radiology healthcare services in Poland in the context of the international and Polish legal provisions concerning the right to healthcare. While funding for imaging studies for patients is widely available and imaging equipment in Poland is improving despite some shortages, the main barrier is identified in the number of specialists capable of assessing the exams. Teleradiology can alleviate this shortage, so the article presents legal provisions and international good practice guidelines in this area, focusing on documents issued by the European Society of Radiology, the American College of Radiology, and the British Royal College of Radiologists. The guidelines concerning such aspects as patients' rights, teleradiologists' qualifications, communication and reporting, responsibility, and technical requirements may help make teleradiology a safe and valuable component of the healthcare system in Poland.

"Crystal memory" Affects the Properties of Peptide Hydrogels - The Case of the Cyclic Tyr-Tyr dipeptide.

In this work a relationship between the crystal form and morphology and rheological properties of peptide-based hydrogels is examined. We show, that under favorable circumstances a correlation between a starting solid material and a self-assembly processes in solution can exist, leading to different properties of a resulting soft matter. This observation, together with an in-depth analysis of the influence of stereochemistry of self-assembled (ll) and (dl) Tyr-Tyr cyclic dipeptides (cYY) on the observed relationship between gelation and crystallization allowed us to gain a deeper understanding of the peptide hydrogelation processes at a molecular level, using liquid state NMR, rheological studies and scanning electron microscopy. In the course of our studies, several crystal forms of (ll)-cYY has been discovered and described in details using single crystal X-ray diffraction, as well as advanced solid state NMR, X-ray diffraction of powders, thermal analysis, FTIR, circular dichroism and crystal structure prediction (CSP) calculations. Subsequently, we found that while (ll)-cYY easily assembles into hydrogels with different properties depending on the starting solid form, (dl)-cYY always precipitated as one crystal form in the tested conditions. Molecular-level justification for this observation is given.

Solid-State Study of the Structure, Dynamics, and Thermal Processes of Safinamide Mesylate─A New Generation Drug for the Treatment of Neurodegenerative Diseases.

Safinamide mesylate (SM), the pure active pharmaceutical ingredient (API) recently used in Parkinson disease treatment, recrystallized employing water-ethanol mixture of solvents (vol/vol 1:9) gives a different crystallographic form compared to SM in Xadago tablets. Pure SM crystallizes as a hemihydrate in the monoclinic system with the P21 space group. Its crystal and molecular structure were determined by means of cryo X-ray crystallography at 100 K. SM in the Xadago tablet exists in anhydrous form in the orthorhombic crystallographic system with the P212121 space group. The water migration and thermal processes in the crystal lattice were monitored by solid-state NMR spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. SM in Xadago in the high-humidity environment undergoes phase transformation to the P21 form which can be easily reversed just by heating up to 80 °C. For the commercial form of the API, there is also a reversible thermal transformation observed between Z' = 1 ↔ Z' = 3 crystallographic forms in the 0-20 °C temperature range. Analysis of molecular motion in the crystal lattice proves that the observed conformational polymorphism is forced by intramolecular dynamics. All above-mentioned processes were analyzed and described employing the NMR crystallography approach with the support of advanced theoretical calculations.

Narrowing down the conformational space with solid-state NMR in crystal structure prediction of linezolid cocrystals.

Many solids crystallize as microcrystalline powders, thus precluding the application of single crystal X-Ray diffraction in structural elucidation. In such cases, a joint use of high-resolution solid-state NMR and crystal structure prediction (CSP) calculations can be successful. However, for molecules showing significant conformational freedom, the CSP-NMR protocol can meet serious obstacles, including ambiguities in NMR signal assignment and too wide conformational search space to be covered by computational methods in reasonable time. Here, we demonstrate a possible way of avoiding these obstacles and making as much use of the two methods as possible in difficult circumstances. In a simple case, our experiments led to crystal structure elucidation of a cocrystal of linezolid (LIN), a wide-range antibiotic, with 2,3-dihydroxybenzoic acid, while a significantly more challenging case of a cocrystal of LIN with 2,4-dihydroxybenzoic acid led to the identification of the most probable conformations of LIN inside the crystal. Having four rotatable bonds, some of which can assume many discreet values, LIN molecule poses a challenge in establishing its conformation in a solid phase. In our work, a set of 27 conformations were used in CSP calculations to yield model crystal structures to be examined against experimental solid-state NMR data, leading to a reliable identification of the most probable molecular arrangements.

New salts of teriflunomide (TFM) - Single crystal X-ray and solid state NMR investigation.

New salts of teriflunomide TFM (drug approved for Multiple Sclerosis treatment) with inorganic counterions: lithium (TFM_Li), sodium (TFM_Na), potassium (TFM_K), rubidium (TFM_Rb), caesium (TFM_Cs) and ammonium (TFM_NH4) were prepared and investigated employing solid state NMR Spectroscopy, Powder X-ray Diffraction PXRD and Single Crystal X-ray Diffraction (SC XRD). Crystal and molecular structures of three salts: TFM_Na (CCDC: 2173257), TFM_Cs (CCDC: 2165288) and TFM_NH4 (CCDC: 2165281) were determined and deposited. Compared to the native TFM, for all crystalline salt structures, a conformational change of the teriflunomide molecule involving about 180-degree rotation of the end group, forming an intramolecular hydrogen bond N-H⋯O is observed. By applying a complementary multi-technique approach, employing 1D and 2D solid state MAS NMR techniques, single and powder X-ray diffraction measurements, as well as the DFT-based GIPAW calculations of NMR chemical shifts for TFM_Na and TFM_Cs allowed to propose structural features of TFM_Li for which it was not possible to obtain adequate material for single crystal X-Ray measurement.

An update on drugs with therapeutic potential for SARS-CoV-2 (COVID-19) treatment.

The COVID-19 pandemic is one of the greatest threats to human health in the 21st century with more than 257 million cases and over 5.17 million deaths reported worldwide (as of November 23, 2021. Various agents were initially proclaimed to be effective against SARS-CoV-2, the etiological agent of COVID-19. Hydroxychloroquine, lopinavir/ritonavir, and ribavirin are all examples of therapeutic agents, whose efficacy against COVID-19 was later disproved. Meanwhile, concentrated efforts of researchers and clinicians worldwide have led to the identification of novel therapeutic options to control the disease including PAXLOVID™ (PF-07321332). Although COVID-19 cases are currently treated using a comprehensive approach of anticoagulants, oxygen, and antibiotics, the novel Pfizer agent PAXLOVID™ (PF-07321332), an investigational COVID-19 oral antiviral candidate, significantly reduced hospitalization time and death rates, based on an interim analysis of the phase 2/3 EPIC-HR (Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients) randomized, double-blind study of non-hospitalized adult patients with COVID-19, who are at high risk of progressing to severe illness. The scheduled interim analysis demonstrated an 89 % reduction in risk of COVID-19-related hospitalization or death from any cause compared to placebo in patients treated within three days of symptom onset (primary endpoint). However, there still exists a great need for the development of additional treatments, as the recommended therapeutic options are insufficient in many cases. Thus far, mRNA and vector vaccines appear to be the most effective modalities to control the pandemic. In the current review, we provide an update on the progress that has been made since April 2020 in clinical trials concerning the effectiveness of therapies available to combat COVID-19. We focus on currently recommended therapeutic agents, including steroids, various monoclonal antibodies, remdesivir, baricitinib, anticoagulants and PAXLOVID™ summarizing the latest original studies and meta-analyses. Moreover, we aim to discuss other currently and previously studied agents targeting COVID-19 that either show no or only limited therapeutic activity. The results of recent studies report that hydroxychloroquine and convalescent plasma demonstrate no efficacy against SARS-CoV-2 infection. Lastly, we summarize the studies on various drugs with incoherent or insufficient data concerning their effectiveness, such as amantadine, ivermectin, or niclosamide.