Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

Low-to-moderate alcohol consumption is associated with increased fibrosis in individuals with metabolic dysfunction-associated steatotic liver disease.

BACKGROUND & AIMS: Both metabolic dysfunction and alcohol consumption cause steatotic liver disease (SLD). The distinction between metabolic dysfunction-associated SLD (MASLD) and MetALD categories is based on arbitrary thresholds of alcohol intake. Thus, we assessed the impact of different levels of alcohol consumption on SLD severity and their interaction with metabolic comorbidities. METHODS: We performed a population-based study with transient elastography (FibroScan®) data from participants in Spain (derivation cohort) and the US (validation cohort). A controlled attenuation parameter ≥275 dB/m was used to define SLD. At least one cardiometabolic risk factor was required to define MASLD. Among patients with MASLD, low alcohol consumption was defined as an average of 5-9 drinks/week, moderate consumption as 10-13 drinks/week for females and 10-20 drinks/week for males, and increased alcohol intake (MetALD) as 14-35 drinks/week for females and 21-42 drinks/week for males. Significant fibrosis was defined as a liver stiffness measurement ≥8 kPa and at-risk metabolic dysfunction-associated steatohepatitis (MASH) as a FAST score ≥0.35. RESULTS: The derivation cohort included 2,227 individuals with MASLD (9% reported low, 14% moderate alcohol consumption) and 76 cases with MetALD. Overall prevalences of significant fibrosis and at-risk MASH were 7.6% and 14.8%, respectively. In the multivariable analysis, alcohol consumption was independently associated with significant fibrosis and at-risk MASH. A dose-dependent increase in the prevalence of significant fibrosis and at-risk MASH was observed between the number of drinks/week and the number of cardiometabolic factors. The validation cohort included 1,732 participants with MASLD, of whom 17% had significant fibrosis and 13% at-risk MASH. This cohort validated the association between moderate intake and MASLD at risk of progression (odds ratio 1.69, 95% CI 1.06-2.71). CONCLUSIONS: Moderate alcohol intake is commonly seen in MASLD and increases the risk of advanced disease to a level similar to that observed in MetALD. IMPACT AND IMPLICATIONS: Metabolic risk factors such as overweight, diabetes or dyslipidemia, and alcohol consumption can cause liver disease. These factors frequently coexist, but their joint effects on liver fibrosis remain uncertain. In this study, we have analyzed individuals from the general population with MASLD (metabolic dysfunction-associated steatotic liver disease) enrolled in Spain and the US. We show that moderate alcohol consumption has a supra-additive effect with metabolic risk factors, exponentially increasing the risk of liver fibrosis. These results suggest that there are no safe limits of daily alcohol intake in patients with unhealthy metabolic status and MASLD.

Budesonide as first-line treatment in patients with autoimmune hepatitis seems inferior to standard predniso(lo)ne administration.

BACKGROUND AND AIMS: In patients with non-severe acute or chronic autoimmune hepatitis (AIH) without cirrhosis, clinical practice guidelines recommend indistinct use of prednisone or budesonide. However, budesonide is infrequently used in clinical practice. We aimed to describe its use and compare its efficacy and safety with prednisone as first-line options. APPROACH AND RESULTS: This was a retrospective, multicenter study of 105 naive AIH patients treated with budesonide as the first-line drug. The control group included 276 patients treated with prednisone. Efficacy was assessed using logistic regression and validated using inverse probability of treatment weighting propensity score. The median time to biochemical response (BR) was 3.1 months in patients treated with budesonide and 4.9 months in those with prednisone. The BR rate was significantly higher in patients treated with prednisone (87% vs. 49% of patients with budesonide, p < 0.001). The probability of achieving BR, assessed using the inverse probability of treatment weighting propensity score, was significantly lower in the budesonide group (OR = 0.20; 95% CI: 0.11-0.38) at any time during follow-up, and at 6 (OR = 0.51; 95% CI: 0.29-0.89) and 12 months after starting treatment (0.41; 95% CI: 0.23-0.73). In patients with transaminases <2 × upper limit of normal, BR was similar in both treatment groups. Prednisone treatment was significantly associated with a higher risk of adverse events (24.2% vs. 15.9%, p = 0.047). CONCLUSIONS: In the real-life setting, the use of budesonide as first-line treatment is low, and it is generally prescribed to patients with perceived less disease activity. Budesonide was inferior to prednisone as a first-line drug but was associated with fewer side effects.

Two SERPINC1 variants affecting N-glycosylation of Asn224 cause severe thrombophilia not detected by functional assays.

Antithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified 2 new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in 4 unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The 2 variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma for 1 hour at 41°C. Moreover, the ß glycoform of the variants, lacking 2 N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant-negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key posttranslational modification that, according to our results, affects folding, secretion, and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Development of the Interosseous Muscles of the Human Hand: Morphological and Functional Aspects of the Terminal Insertion.

INTRODUCTION: To date, there have been no studies conducted on the development of interosseous muscles (IO) in the human hand. This study aimed to investigate the development of these muscles in order to clarify their terminal insertions and their relationship with the metacarpophalangeal joints. METHODS: Serial sections of 25 human specimens (9 embryos and 16 fetuses) between the 7th and 14th weeks of development, sourced from the Collection of the Department of Anatomy and Embryology at UCM Faculty of Medicine, were analyzed bilaterally using a conventional optical microscope. RESULTS: Our findings revealed that, during the 7th week of development, the metacarpophalangeal interzone mesenchyme extended into the extensor apparatus of the fingers. Furthermore, we observed that the joint capsule and the tendon of the IO derive from the articular interzone mesenchyme. By the end of the 7th week, corresponding to Carnegie stage 21, the myotendinous junction appeared, initiating cavitation of the metacarpophalangeal joint. During the fetal period, the terminal insertions of the IO were identified: both the dorsal interosseous (DI) and palmar interosseous (PI) muscles insert into the metacarpophalangeal joint capsule and establish a connection with the volar plate located at the base of the proximal phalanx and the extensor apparatus. Some muscle fibers also attach to the joint capsule at the level of the proximal synovial cul-de-sac. The functional implications of these findings are discussed within this work. CONCLUSION: This study provides the first detailed description of the development of the interosseous muscles in the human hand.

Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

2023 updated MASCC/ESMO consensus recommendations: controlling nausea and vomiting with chemotherapy of low or minimal emetic potential.

PURPOSE: Review the literature to update the MASCC guidelines from 2016 for controlling nausea and vomiting with systemic cancer treatment of low and minimal emetic potential. METHODS: A working group performed a systematic literature review using Medline, Embase, and Scopus databases between June 2015 and January 2023 of the management of antiemetic prophylaxis for anticancer therapy of low or minimal emetic potential. A consensus committee reviewed recommendations and required a consensus of 67% or greater and a change in outcome of at least 10%. RESULTS: Of 293 papers identified, 15 had information about managing systemic cancer treatment regimens of low or minimal emetic potential and/or compliance with previous management recommendations. No new evidence was reported that would change the current MASCC recommendations. No antiemetic prophylaxis is recommended for minimal emetic potential therapy, and single agents recommended for low emetic potential chemotherapy for acute emesis, but no prophylaxis is recommended for delayed emesis. Commonly, rescue medication includes antiemetics prescribed for the next higher level of emesis. CONCLUSION: There is insufficient data to change the current guidelines. Future studies should seek to more accurately determine the risk of emesis with LEC beyond the emetogenicity of the chemotherapy to include patient-related risk assessment.

Innovative Therapeutic Approaches in Non-Alcoholic Fatty Liver Disease: When Knowing Your Patient Is Key.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of disorders ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic steatosis may result from the dysfunction of multiple pathways and thus multiple molecular triggers involved in the disease have been described. The development of NASH entails the activation of inflammatory and fibrotic processes. Furthermore, NAFLD is also strongly associated with several extra-hepatic comorbidities, i.e., metabolic syndrome, type 2 diabetes mellitus, obesity, hypertension, cardiovascular disease and chronic kidney disease. Due to the heterogeneity of NAFLD presentations and the multifactorial etiology of the disease, clinical trials for NAFLD treatment are testing a wide range of interventions and drugs, with little success. Here, we propose a narrative review of the different phenotypic characteristics of NAFLD patients, whose disease may be triggered by different agents and driven along different pathophysiological pathways. Thus, correct phenotyping of NAFLD patients and personalized treatment is an innovative therapeutic approach that may lead to better therapeutic outcomes.

Faecal Microbiota Transplantation, Paving the Way to Treat Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is currently the most prevalent cause of chronic liver disease (CLD). Currently, the only therapeutic recommendation available is a lifestyle change. However, adherence to this approach is often difficult to guarantee. Alteration of the microbiota and an increase in intestinal permeability seem to be key in the development and progression of NAFLD. Therefore, the manipulation of microbiota seems to provide a promising therapeutic strategy. One way to do so is through faecal microbiota transplantation (FMT). Here, we summarize the key aspects of FMT, detail its current indications and highlight the most recent advances in NAFLD.

Fatty Liver Disease, Metabolism and Alcohol Interplay: A Comprehensive Review.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, and its incidence has been increasing in recent years because of the high prevalence of obesity and metabolic syndrome in the Western population. Alcohol-related liver disease (ArLD) is the most common cause of cirrhosis and constitutes the leading cause of cirrhosis-related deaths worldwide. Both NAFLD and ArLD constitute well-known causes of liver damage, with some similarities in their pathophysiology. For this reason, they can lead to the progression of liver disease, being responsible for a high proportion of liver-related events and liver-related deaths. Whether ArLD impacts the prognosis and progression of liver damage in patients with NAFLD is still a matter of debate. Nowadays, the synergistic deleterious effect of obesity and diabetes is clearly established in patients with ArLD and heavy alcohol consumption. However, it is still unknown whether low to moderate amounts of alcohol are good or bad for liver health. The measurement and identification of the possible synergistic deleterious effect of alcohol consumption in the assessment of patients with NAFLD is crucial for clinicians, since early intervention, advising abstinence and controlling cardiovascular risk factors would improve the prognosis of patients with both comorbidities. This article seeks to perform a comprehensive review of the pathophysiology of both disorders and measure the impact of alcohol consumption in patients with NAFLD.

Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency.

Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in SERPINC1 in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.

Factor XI in Carriers of Antiphospholipid Antibodies: Elevated Levels Associated with Symptomatic Thrombotic Cases, While Low Levels Linked to Asymptomatic Cases.

Antiphospholipid syndrome (APS) is a thromboinflammatory disorder caused by circulating antiphospholipid autoantibodies (aPL) and characterized by an increased risk of thrombotic events. The pathogenic mechanisms of these antibodies are complex and not fully understood, but disturbances in coagulation and fibrinolysis have been proposed to contribute to the thrombophilic state. This study aims to evaluate the role of an emerging hemostatic molecule, FXI, in the thrombotic risk of patients with aPL. Cross-sectional and observational study of 194 consecutive and unrelated cases with aPL recruited in a single center: 82 asymptomatic (AaPL) and 112 with primary antiphospholipid syndrome (APS). Clinical and epidemiological variables were collected. The profile of aPL was determined. Plasma FXI was evaluated by Western blotting and two coagulation assays (FXI:C). In cases with low FXI, molecular analysis of the F11 gene was performed. FXI:C levels were significantly higher in patients with APS than in patients with AaPL (122.8 ± 33.4 vs. 104.5 ± 27.5; p < 0.001). Multivariate analysis showed a significant association between symptomatic patients with aPL (APS) and high FXI (>150%) (OR = 11.57; 95% CI: 1.47-90.96; p = 0.020). In contrast, low FXI (<70%), mostly caused by inhibitors, was less frequent in the group of patients with APS compared to AaPL (OR = 0.17; 95%CI: 0.36-0.86; p = 0.032). This study suggests that FXI levels may play a causal role in the prothrombotic state induced by aPLs and holds the promise of complementary treatments in APS patients by targeting FXI.

Environmentally friendly starch/alginate aerogels for copper adsorption from aqueous media. A microstructural and kinetic study.

This work investigated the synthesis and characterization of alginate/starch porous materials and their application as copper ions adsorbents from aqueous media. Initially, pregel aqueous solutions with different biopolymer concentrations (1, 3, and 5% w/w) and alginate contents (25, 50, and 75% w/w) were prepared. Hydrogel formation was performed by internal and external gelation methods. Finally, the drying step was done via CO2sc leading to aerogels and via freeze-drying leading to cryogels. Process parameters influence on the final properties of materials was evaluated by BET isotherms, SEM, EDS, and TGA. Regardless the gelation method applied, interesting materials with meso- and macro-pore structure were prepared from pregel mixtures with 3% w/w biopolymer concentration and an alginate content of only 25% w/w. Low alginate content reduces the final cost of the materials. Concerning copper removal, the adsorption data were well fitted to the pseudo-second order kinetic model for aerogels and cryogels, showing aerogels the highest adsorption capacity (40 mg/g) and removal efficiency (∼ 92%). Materials demonstrated excellent reusability throughout five consecutive adsorption/desorption cycles. Hence, environmentally friendly materials with a high practical value as low-cost bioadsorbents were synthesized, having great performance in the removal of copper ions from aqueous solution.

Dissecting the transcriptional program of phosphomannomutase 2-deficient cells: Lymphoblastoide B cell lines as a valuable model for congenital disorders of glycosylation studies.

Congenital disorders of glycosylation (CDG) include 150 genetically and clinically heterogeneous diseases, showing significant glycoprotein hypoglycosylation that leads to pathological consequences in multiple organs and systems whose underlying mechanisms are not yet understood. A few cellular and animal models have been used to study specific CDG characteristics, although they have given limited information due to the few CDG mutations tested and the still missing comprehensive molecular and cellular basic research. Here, we provide specific gene expression profiles, based on ribonucleic acid (RNA) microarray analysis, together with some biochemical and cellular characteristics of a total of nine control Epstein-Barr virus-transformed lymphoblastoid B cell lines (B-LCL) and 13 CDG B-LCL from patients carrying severe mutations in the phosphomannomutase 2 (PMM2) gene, strong serum protein hypoglycosylation and neurological symptoms. Significantly dysregulated genes in PMM2-CDG cells included those regulating stress responses, transcription factors, glycosylation, motility, cell junction and, importantly, those related to development and neuronal differentiation and synapse, such as carbonic anhydrase 2 (CA2) and ADAM23. PMM2-CDG-associated biological consequences involved the unfolded protein response, RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Changes in the transcriptional and CA2 protein levels are consistent with the CDG physiopathology. These results demonstrate the global transcriptional impact in phosphomannomutase 2-deficient cells, reveal CA2 as a potential cellular biomarker and confirm B-LCL as an advantageous model for CDG studies.

Molecular and clinical characterization of transient antithrombin deficiency: A new concept in congenital thrombophilia.

Antithrombin deficiency, the most severe thrombophilia, might be underestimated, since it is only investigated in cases with consistent functional deficiency or family history. We have analyzed 444 consecutive, unrelated cases, from 1998 to 2021, with functional results supporting antithrombin deficiency in at least one sample. Plasma antithrombin was evaluated by functional and biochemical methods in at least two samples. SERPINC1 gene was analyzed by sequencing and MPLA. Hypoglycosylation was studied by electrophoresis and high-performance liquid chromatography (HPLC). In 260 of 305 cases (85.2%) with constitutive deficiency (activity < 80% in all samples), a SERPINC1 (N = 250), or N-glycosylation defect (N = 10) was observed, while 45 remained undetermined. The other 139 cases had normal antithrombin activity (≥ 80%) in at least one sample, what we called transient deficiency. Sixty-one of these cases (43.9%) had molecular defects: 48 had SERPINC1 variants, with two recurrent mutations (p.Ala416Ser[Cambridge II], N = 15; p.Val30Glu[Dublin], N = 12), and 13 hypoglycosylation. Thrombotic complications occurred in transient deficiency, but were less frequent, latter-onset, and had a higher proportion of arterial events than in constitutive deficiency. Two mechanisms explained transient deficiency: The limitation of functional methods to detect some variants and the influence of external factors on the pathogenic consequences of these mutations. Our study reveals a molecular defect in a significant proportion of cases with transient antithrombin deficiency, and changes the paradigm of thrombophilia, as the pathogenic effect of some mutations might depend on external factors and be present only at certain timepoints. Antithrombin deficiency is underestimated, and molecular screening might be appropriate in cases with fluctuating laboratory findings.

Latin American Consensus on the Management of Sepsis in Children: Sociedad Latinoamericana de Cuidados Intensivos Pediátricos [Latin American Pediatric Intensive Care Society] (SLACIP) Task Force: Executive Summary.

Objective: The aim of this study was to develop evidence-based recommendations for the diagnosis and treatment of sepsis in children in low- and middle-income countries (LMICs), more specifically in Latin America. Design: A panel was formed consisting of 27 experts with experience in the treatment of pediatric sepsis and two methodologists working in Latin American countries. The experts were organized into 10 nominal groups, each coordinated by a member. Methods: A formal consensus was formed based on the modified Delphi method, combining the opinions of nominal groups of experts with the interpretation of available scientific evidence, in a systematic process of consolidating a body of recommendations. The systematic search was performed by a specialized librarian and included specific algorithms for the Cochrane Specialized Register, PubMed, Lilacs, and Scopus, as well as for OpenGrey databases for grey literature. The GRADEpro GDT guide was used to classify each of the selected articles. Special emphasis was placed on search engines that included original research conducted in LMICs. Studies in English, Spanish, and Portuguese were covered. Through virtual meetings held between February 2020 and February 2021, the entire group of experts reviewed the recommendations and suggestions. Result: At the end of the 12 months of work, the consensus provided 62 recommendations for the diagnosis and treatment of pediatric sepsis in LMICs. Overall, 60 were strong recommendations, although 56 of these had a low level of evidence. Conclusions: These are the first consensus recommendations for the diagnosis and management of pediatric sepsis focused on LMICs, more specifically in Latin American countries. The consensus shows that, in these regions, where the burden of pediatric sepsis is greater than in high-income countries, there is little high-level evidence. Despite the limitations, this consensus is an important step forward for the diagnosis and treatment of pediatric sepsis in Latin America.

Comparative Physical Study of Three Pharmaceutically Active Benzodiazepine Derivatives: Crystalline versus Amorphous State and Crystallization Tendency.

Chemical derivatization and amorphization are two possible strategies to improve the solubility and bioavailability of drugs, which is a key issue for the pharmaceutical industry. In this contribution, we explore whether both strategies can be combined by studying how small differences in the molecular structure of three related pharmaceutical compounds affect their crystalline structure and melting point (Tm), the relaxation dynamics in the amorphous phase, and the glass transition temperature (Tg), as well as the tendency toward recrystallization. Three benzodiazepine derivatives of almost same molecular mass and structure (Diazepam, Nordazepam and Tetrazepam) were chosen as model compounds. Nordazepam is the only one that displays N-H···O hydrogen bonds both in crystalline and amorphous phases, which leads to a significantly higher Tm (by 70-80 K) and Tg (by 30-40 K) compared to those of Tetrazepam and Diazepam (which display similar values of characteristic temperatures). The relaxation dynamics in the amorphous phase, as determined experimentally using broadband dielectric spectroscopy, is dominated by a structural relaxation and a Johari-Goldstein secondary relaxation, both of which scale with the reduced temperature T/Tg. The kinetic fragility index is very low and virtually the same (mp ≈ 32) in all three compounds. Two more secondary relaxations are observed in the glass state: the slower of the two has virtually the same relaxation time and activation energy in all three compounds, and is assigned to the inter-enantiomer conversion dynamics of the flexible diazepine heterocycle between isoenergetic P and M conformations. We tentatively assign the fastest secondary relaxation, present only in Diazepam and Tetrazepam, to the rigid rotation of the fused diazepine-benzene double ring relative to the six-membered carbon ring. Such motion appears to be largely hindered in glassy Nordazepam, possibly due to the presence of the hydrogen bonds. Supercooled liquid Tetrazepam and Nordazepam are observed to crystallize into their stable crystalline form with an Avrami exponent close to unity indicating unidimensional growth with only sporadic nucleation, which allows a direct assessment of the crystal growth rate. Despite the very similar growth mode, the two derivatives exhibit very different kinetics for a fixed value of the reduced temperature and thus of the structural relaxation time, with Nordazepam displaying slower growth kinetics. Diazepam does not instead display any tendency toward recrystallization over short periods of time (even close to Tm). Both these observations in three very similar diazepine derivatives provide direct evidence that the kinetics of recrystallization of amorphous pharmaceuticals is not a universal function, at least in the supercooled liquid phase, of the structural or the conformational relaxation dynamics, and it is not simply correlated with related parameters such as the kinetic fragility or activation barrier of the structural relaxation. Only the crystal growth rate, and not the nucleation rate, shows a correlation with the presence or absence of hydrogen bonding.

High penetrance of inferior vena cava system atresia in severe thrombophilia caused by homozygous antithrombin Budapest 3 variant: Description of a new syndrome.

Atresia of inferior vena cava (IVC) is a rare congenital malformation associated with high risk of venous thrombosis that still has unknown etiology, although intrauterine IVC thrombosis has been suggested to be involved. The identification of IVC atresia in a case with early idiopathic venous thrombosis and antithrombin deficiency caused by the homozygous SERPINC1 c.391C > T variant (p.Leu131Phe; antithrombin Budapest 3) encouraged us to evaluate the role of this severe thrombophilia in this vascular abnormality. We have done a cross-sectional study in previously identified cohorts of patients homozygous for the Budapest 3 variant (N = 61) selected from 1118 patients with congenital antithrombin deficiency identified in two different populations: Spain (N = 692) and Hungary (N = 426). Image analysis included computed tomography and phlebography. Atresia of the IVC system was observed in 17/24 cases (70.8%, 95% confidence interval [CI]: 48.9%-87.3%) homozygous for antithrombin Budapest 3 with available computed tomography (5/8 and 12/16 in the Spanish and Hungarian cohorts, respectively), 16 had an absence of infrarenal IVC and one had atresia of the left common iliac vein. All cases with vascular defects had compensatory mechanisms, azygos-hemiazygos continuation or double IVC, and seven also had other congenital anomalies. Short tandem repeat analysis supported the specific association of the IVC system atresia with SERPINC1. We show the first evidence of the association of a severe thrombophilia with IVC system atresia, supporting the possibility that a thrombosis in the developing fetal vessels is the reason for this anomaly. Our hypothesis-generating results encourage further studies to investigate severe thrombophilic states in patients with atresia of IVC.

Feasibility of a hybrid clinical trial for respiratory virus detection in toddlers during the influenza season.

BACKGROUND: Traditional clinical trials are conducted at investigator sites. Participants must visit healthcare facilities several times for the trial procedures. Decentralized clinical trials offer an interesting alternative. They use telemedicine and other technological solutions (apps, monitoring devices or web platforms) to decrease the number of visits to study sites, minimise the impact on daily routine, and decrease geographical barriers for participants. Not much information is available on the use of decentralization in randomized clinical trials with vaccines. METHODS: A hybrid clinical trial may be assisted by parental recording of symptoms using electronic log diaries in combination with home collected nasal swabs. During two influenza seasons, children aged 12 to 35 months with a history of recurrent acute respiratory infections were recruited in 12 primary health centers of the Valencia Region in Spain. Parents completed a symptom diary through an ad hoc mobile app that subsequently assessed whether it was an acute respiratory infection and requested collection of a nasal swab. Feasibility was measured using the percentage of returned electronic diaries and the validity of nasal swabs collected during the influenza season. Respiratory viruses were detected by real-time PCR. RESULTS: Ninety-nine toddlers were enrolled. Parents completed 10,476 electronic diaries out of the 10,804 requested (97%). The mobile app detected 188 potential acute respiratory infections (ARIs) and requested a nasal swab. In 173 (92%) ARI episodes a swab was taken. 165 (95.4%) of these swabs were collected at home and 144 (87.3%) of them were considered valid for laboratory testing. Overall, 152 (81%) of the ARIs detected in the study had its corresponding valid sample collected. CONCLUSIONS: Hybrid procedures used in this clinical trial with the influenza vaccine in toddlers were considered adequate, as we diagnosed most of the ARI cases on time, and had a valid swab in 81% of the cases. Hybrid clinical trials improve participant adherence to the study procedures and could improve recruitment and quality of life of the participants and the research team by decreasing the number of visits to the investigator site. This report emphasises that the conduct of hybrid CTs is a valid alternative to traditional CTs with vaccines. This hybrid CT achieved high adherence of participant to the study procedures. TRIAL REGISTRATION: 2019-001186-33 (EudraCT).

N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.