Persistent and fatal severe acute respiratory syndrome coronavirus 2 infection in a patient with severe hypogammaglobulinemia: a case report.

BACKGROUND: Viruses are constantly changing as a result of mutations, and new viral variants are expected to appear over time. The virus that causes coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2, is not excluded from this condition. Patients with some types of immunodeficiency have been reported to experience symptoms that vary from mild to severe, or even death, after being infected with severe acute respiratory syndrome coronavirus 2. We report a case of a woman with severe hypogammaglobulinemia who developed a prolonged and fatal severe acute respiratory syndrome coronavirus 2 infection. CASE PRESENTATION: A 60-year-old mestizo female with a previous history of severe hypogammaglobulinemia manifested by recurrent pulmonary infections and follicular bronchiolitis. She received a monthly treatment of intravenous immunoglobulins and was admitted after report of a neurological manifestation related to a left thalamic inflammatory lesion, for a duration of 2 weeks of hospitalization, indicated for the study of her neurological condition, including brain biopsy. Both on admission and 1 week later, nasopharyngeal polymerase chain reaction tests for severe acute respiratory syndrome coronavirus 2 were performed and reported negative. In the third week of hospitalization, she developed pulmonary symptoms, and a positive test result for severe acute respiratory syndrome coronavirus 2 was evidenced. On Day 3, the patients' condition worsened as the infection progressed to respiratory failure and required mechanical ventilation. On Day 8 after the coronavirus disease 2019 diagnosis, the polymerase chain reaction test for severe acute respiratory syndrome coronavirus 2 showed persistent detection of the virus. Various bacterial coinfections, including Klebsiella pneumoniae and Enterobacter cloacae, were diagnosed and treated. On Day 35, her pulmonary symptoms worsened, and the results of the severe acute respiratory syndrome coronavirus 2 polymerase chain reaction test remained positive. On Day 36, despite all the respiratory support, the patient died. The severe acute respiratory syndrome coronavirus 2 virus was sequenced at the beginning and 8 days after the onset of the disease, and the strain, without obvious mutations in the gene that encodes spike protein, was identified. CONCLUSIONS: This clinical case showed persistent severe acute respiratory syndrome coronavirus 2 detection after 35 days of infection in a patient with severe hypogammaglobulinemia. The sequencing of the virus showed no mutations on the spike protein at 8 days, indicating that, in this case, the persistence of the viral detection was associated with immunodeficiency instead of changes in the viral components.

Pregnancy-specific responses to COVID-19 revealed by high-throughput proteomics of human plasma.

BACKGROUND: Pregnant women are at greater risk of adverse outcomes, including mortality, as well as obstetrical complications resulting from COVID-19. However, pregnancy-specific changes that underlie such worsened outcomes remain unclear. METHODS: Plasma samples were collected from pregnant women and non-pregnant individuals (male and female) with (n = 72 pregnant, 52 non-pregnant) and without (n = 29 pregnant, 41 non-pregnant) COVID-19. COVID-19 patients were grouped as asymptomatic, mild, moderate, severe, or critically ill according to NIH classifications. Proteomic profiling of 7,288 analytes corresponding to 6,596 unique protein targets was performed using the SOMAmer platform. RESULTS: Herein, we profile the plasma proteome of pregnant and non-pregnant COVID-19 patients and controls and show alterations that display a dose-response relationship with disease severity; yet, such proteomic perturbations are dampened during pregnancy. In both pregnant and non-pregnant state, the proteome response induced by COVID-19 shows enrichment of mediators implicated in cytokine storm, endothelial dysfunction, and angiogenesis. Shared and pregnancy-specific proteomic changes are identified: pregnant women display a tailored response that may protect the conceptus from heightened inflammation, while non-pregnant individuals display a stronger response to repel infection. Furthermore, the plasma proteome can accurately identify COVID-19 patients, even when asymptomatic or with mild symptoms. CONCLUSION: This study represents the most comprehensive characterization of the plasma proteome of pregnant and non-pregnant COVID-19 patients. Our findings emphasize the distinct immune modulation between the non-pregnant and pregnant states, providing insight into the pathogenesis of COVID-19 as well as a potential explanation for the more severe outcomes observed in pregnant women.

Pregnant COVID-19 patients are at increased risk of experiencing complications and severe outcomes compared to the general population. However, the reasons for this heightened risk are still unclear. We measured the proteins present in the blood of pregnant and non-pregnant patients with COVID-19 and compared these to healthy individuals. We found that some COVID-19-associated proteins were present at lower levels in pregnant women, which could help to protect the fetus from harmful inflammation, the body's natural response to infection. While some proteins affected by COVID-19 are shared between pregnant and non-pregnant patients, others were distinctly affected only in pregnant women, providing a potential explanation for the more severe outcomes in this group.

Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil / Diversidade de fungos em morcegos de fragmentos florestais urbanos de Sinop, Mato Grosso, Brasil

Bats are important for the homeostasis of ecosystems and serve as hosts of various microorganisms including bacteria, viruses, and fungi with pathogenic potential. This study aimed to isolate fungi from biological samples obtained from bats captured in the city of Sinop (state of Mato Grosso, Brazil), where large areas of deforestation exist due to urbanization and agriculture. On the basis of the flow of people and domestic animals, 48 bats were captured in eleven urban forest fragments. The samples were processed and submitted to microbiological cultures, to isolate and to identify the fungal genera. Thirty-four (70.83%) of the captured bats were positive for fungi; 18 (37.5%) and 16 (33.33%) of these bats were female and male, respectively. Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp., and Candida sp., which may cause opportunistic infections, were isolated. The bat species with the highest number of fungal isolates was Molossus molossus: 21 isolates (43.8%). According to our results, bats captured in urban forest fragments in Sinop harbor pathogenic fungi, increasing the risk of opportunistic fungal infections in humans and domestic animals.

Os morcegos apresentam grande importância na homeostasia dos ecossistemas e são hospedeiros de uma rica diversidade de micro-organismos como bactérias, vírus e fungos com potencial patogênico. Portanto, este estudo visou isolar fungos presentes em amostras biológicas de morcegos na cidade de Sinop - MT, que possui grandes áreas de desmatamento devido à urbanização e agricultura. Foram capturados 48 morcegos de diferentes espécies, em onze fragmentos florestais urbanos definidos de acordo com fluxo de pessoas e animais domésticos, para obtenção de amostras biológicas. Essas amostras foram processadas e submetidas aos cultivos microbiológicos, para isolamento e identificação dos gêneros dos fungos. Dos 48 morcegos, 34 (70,83%) foram positivos para pelos menos um gênero de fungo, sendo 18 (37,5%) fêmeas e 16 (33,33%) machos, e os gêneros isolados a partir das amostras biológicas foram Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp. e Candida sp., que podem ser causadores de infecções oportunistas. Desse total, a espécie que apresentou maior positividade para pelo menos um gênero de fungo foi Molossus molossus com 21 (43,8%). Nossos resultados demonstram que os morcegos capturados nos fragmentos florestais urbanos na cidade de Sinop - MT, podem atuar como agentes veiculadores de fungos com potencial patogênico, aumentando assim o risco de exposição e aquisição de infecções fúngicas oportunistas por pessoas e animais domésticos.

Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil

Abstract Bats are important for the homeostasis of ecosystems and serve as hosts of various microorganisms including bacteria, viruses, and fungi with pathogenic potential. This study aimed to isolate fungi from biological samples obtained from bats captured in the city of Sinop (state of Mato Grosso, Brazil), where large areas of deforestation exist due to urbanization and agriculture. On the basis of the flow of people and domestic animals, 48 bats were captured in eleven urban forest fragments. The samples were processed and submitted to microbiological cultures, to isolate and to identify the fungal genera. Thirty-four (70.83%) of the captured bats were positive for fungi; 18 (37.5%) and 16 (33.33%) of these bats were female and male, respectively. Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp., and Candida sp., which may cause opportunistic infections, were isolated. The bat species with the highest number of fungal isolates was Molossus molossus: 21 isolates (43.8%). According to our results, bats captured in urban forest fragments in Sinop harbor pathogenic fungi, increasing the risk of opportunistic fungal infections in humans and domestic animals.

Resumo Os morcegos apresentam grande importância na homeostasia dos ecossistemas e são hospedeiros de uma rica diversidade de micro-organismos como bactérias, vírus e fungos com potencial patogênico. Portanto, este estudo visou isolar fungos presentes em amostras biológicas de morcegos na cidade de Sinop - MT, que possui grandes áreas de desmatamento devido à urbanização e agricultura. Foram capturados 48 morcegos de diferentes espécies, em onze fragmentos florestais urbanos definidos de acordo com fluxo de pessoas e animais domésticos, para obtenção de amostras biológicas. Essas amostras foram processadas e submetidas aos cultivos microbiológicos, para isolamento e identificação dos gêneros dos fungos. Dos 48 morcegos, 34 (70,83%) foram positivos para pelos menos um gênero de fungo, sendo 18 (37,5%) fêmeas e 16 (33,33%) machos, e os gêneros isolados a partir das amostras biológicas foram Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp. e Candida sp., que podem ser causadores de infecções oportunistas. Desse total, a espécie que apresentou maior positividade para pelo menos um gênero de fungo foi Molossus molossus com 21 (43,8%). Nossos resultados demonstram que os morcegos capturados nos fragmentos florestais urbanos na cidade de Sinop - MT, podem atuar como agentes veiculadores de fungos com potencial patogênico, aumentando assim o risco de exposição e aquisição de infecções fúngicas oportunistas por pessoas e animais domésticos.

Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil / Diversidade de fungos em morcegos de fragmentos florestais urbanos de Sinop, Mato Grosso, Brasil

Abstract Bats are important for the homeostasis of ecosystems and serve as hosts of various microorganisms including bacteria, viruses, and fungi with pathogenic potential. This study aimed to isolate fungi from biological samples obtained from bats captured in the city of Sinop (state of Mato Grosso, Brazil), where large areas of deforestation exist due to urbanization and agriculture. On the basis of the flow of people and domestic animals, 48 bats were captured in eleven urban forest fragments. The samples were processed and submitted to microbiological cultures, to isolate and to identify the fungal genera. Thirty-four (70.83%) of the captured bats were positive for fungi; 18 (37.5%) and 16 (33.33%) of these bats were female and male, respectively. Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp., and Candida sp., which may cause opportunistic infections, were isolated. The bat species with the highest number of fungal isolates was Molossus molossus: 21 isolates (43.8%). According to our results, bats captured in urban forest fragments in Sinop harbor pathogenic fungi, increasing the risk of opportunistic fungal infections in humans and domestic animals.

Resumo Os morcegos apresentam grande importância na homeostasia dos ecossistemas e são hospedeiros de uma rica diversidade de micro-organismos como bactérias, vírus e fungos com potencial patogênico. Portanto, este estudo visou isolar fungos presentes em amostras biológicas de morcegos na cidade de Sinop - MT, que possui grandes áreas de desmatamento devido à urbanização e agricultura. Foram capturados 48 morcegos de diferentes espécies, em onze fragmentos florestais urbanos definidos de acordo com fluxo de pessoas e animais domésticos, para obtenção de amostras biológicas. Essas amostras foram processadas e submetidas aos cultivos microbiológicos, para isolamento e identificação dos gêneros dos fungos. Dos 48 morcegos, 34 (70,83%) foram positivos para pelos menos um gênero de fungo, sendo 18 (37,5%) fêmeas e 16 (33,33%) machos, e os gêneros isolados a partir das amostras biológicas foram Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp. e Candida sp., que podem ser causadores de infecções oportunistas. Desse total, a espécie que apresentou maior positividade para pelo menos um gênero de fungo foi Molossus molossus com 21 (43,8%). Nossos resultados demonstram que os morcegos capturados nos fragmentos florestais urbanos na cidade de Sinop - MT, podem atuar como agentes veiculadores de fungos com potencial patogênico, aumentando assim o risco de exposição e aquisição de infecções fúngicas oportunistas por pessoas e animais domésticos.

Pregnancy-specific responses to COVID-19 are revealed by high-throughput proteomics of human plasma.

Pregnant women are at greater risk of adverse outcomes, including mortality, as well as obstetrical complications resulting from COVID-19. However, pregnancy-specific changes that underlie such worsened outcomes remain unclear. Herein, we profiled the plasma proteome of pregnant and non-pregnant COVID-19 patients and controls and showed alterations that display a dose-response relationship with disease severity; yet, such proteomic perturbations are dampened during pregnancy. In both pregnant and non-pregnant state, the proteome response induced by COVID-19 showed enrichment of mediators implicated in cytokine storm, endothelial dysfunction, and angiogenesis. Shared and pregnancy-specific proteomic changes were identified: pregnant women display a tailored response that may protect the conceptus from heightened inflammation, while non-pregnant individuals display a stronger response to repel infection. Furthermore, the plasma proteome can accurately identify COVID-19 patients, even when asymptomatic or with mild symptoms. This study represents the most comprehensive characterization of the plasma proteome of pregnant and non-pregnant COVID-19 patients.

Genomic characterization of SARS-CoV-2 and its association with clinical outcomes: a 1-year longitudinal study of the pandemic in Colombia.

OBJECTIVES: This study aimed to explore associations between the molecular characterization of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and disease severity in ambulatory and hospitalized patients in two main Colombian epicentres during the first year of the coronavirus disease 2019 pandemic. METHODS: In total, 1000 patients with SARS-CoV-2 infection were included in this study. Clinical data were collected from 997 patients, and 678 whole-genome sequences were obtained by massively parallel sequencing. Bivariate, multi-variate, and classification and regression tree analyses were run between clinical and genomic variables. RESULTS: Age >88 years, and infection with lineages B.1.1, B.1.1.388, B.1.523 or B.1.621 for patients aged 71-88 years were associated with death [odds ratio (OR) 6.048036, 95% confidence interval (CI) 1.346567-32.92521; P=0.01718674]. The need for hospitalization was associated with higher age and comorbidities. The hospitalization rate increased significantly for patients aged 38-51 years infected with lineages A, B, B.1.1.388, B.1.1.434, B.1.153, B.1.36.10, B.1.411, B.1.471, B.1.558 or B.1.621 (OR 8.368427, 95% CI 2.573145-39.10672, P=0.00012). Associations between clades and clinical outcomes diverged from previously reported data. CONCLUSIONS: Infection with lineage B.1.621 increased the hospitalization and mortality rates. These findings, plus the rapidly increasing prevalence in Colombia and other countries, suggest that lineage B.1.621 should be considered as a 'variant of interest'. If associated disease severity is confirmed, possible designation as a 'variant of concern' should be considered.

Mutation profile of SARS-CoV-2 genome in a sample from the first year of the pandemic in Colombia.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the etiopathogenic agent of COVID-19, a condition that has led to a formally recognized pandemic by March 2020 (World Health Organization -WHO). The SARS-CoV-2 genome is constituted of 29,903 base pairs, that code for four structural proteins (N, M, S, and E) and more than 20 non-structural proteins. Mutations in any of these regions, especially in those that encode for the structural proteins, have allowed the identification of diverse lineages around the world, some of them named as Variants of Concern (VOC) and Variants of Interest (VOI), according to the WHO and CDC. In this study, by using Next Generation Sequencing (NGS) technology, we sequenced the SARS-CoV-2 genome of 422 samples from Colombian residents, all of them collected between April 2020 and January 2021. We obtained genetic information from 386 samples, leading us to the identification of 14 new lineages circulating in Colombia, 13 of which were identified for the first time in South America. GH was the predominant GISAID clade in our sample. Most mutations were either missense (53.6%) or synonymous mutations (37.4%), and most genetic changes were located in the ORF1ab gene (63.9%), followed by the S gene (12.9%). In the latter, we identified mutations E484K, L18F, and D614G. Recent evidence suggests that these mutations concede important particularities to the virus, compromising host immunity, the diagnostic test performance, and the effectiveness of some vaccines. Some important lineages containing these mutations are the Alpha, Beta, and Gamma (WHO Label). Further genomic surveillance is important for the understanding of emerging genomic variants and their correlation with disease severity.

Effect of High-Flow Oxygen Therapy vs Conventional Oxygen Therapy on Invasive Mechanical Ventilation and Clinical Recovery in Patients With Severe COVID-19: A Randomized Clinical Trial.

IMPORTANCE: The effect of high-flow oxygen therapy vs conventional oxygen therapy has not been established in the setting of severe COVID-19. OBJECTIVE: To determine the effect of high-flow oxygen therapy through a nasal cannula compared with conventional oxygen therapy on need for endotracheal intubation and clinical recovery in severe COVID-19. DESIGN, SETTING, AND PARTICIPANTS: Randomized, open-label clinical trial conducted in emergency and intensive care units in 3 hospitals in Colombia. A total of 220 adults with respiratory distress and a ratio of partial pressure of arterial oxygen to fraction of inspired oxygen of less than 200 due to COVID-19 were randomized from August 2020 to January 2021, with last follow-up on February 10, 2021. INTERVENTIONS: Patients were randomly assigned to receive high-flow oxygen through a nasal cannula (n = 109) or conventional oxygen therapy (n = 111). MAIN OUTCOMES AND MEASURES: The co-primary outcomes were need for intubation and time to clinical recovery until day 28 as assessed by a 7-category ordinal scale (range, 1-7, with higher scores indicating a worse condition). Effects of treatments were calculated with a Cox proportional hazards model adjusted for hypoxemia severity, age, and comorbidities. RESULTS: Among 220 randomized patients, 199 were included in the analysis (median age, 60 years; n = 65 women [32.7%]). Intubation occurred in 34 (34.3%) randomized to high-flow oxygen therapy and in 51 (51.0%) randomized to conventional oxygen therapy (hazard ratio, 0.62; 95% CI, 0.39-0.96; P = .03). The median time to clinical recovery within 28 days was 11 (IQR, 9-14) days in patients randomized to high-flow oxygen therapy vs 14 (IQR, 11-19) days in those randomized to conventional oxygen therapy (hazard ratio, 1.39; 95% CI, 1.00-1.92; P = .047). Suspected bacterial pneumonia occurred in 13 patients (13.1%) randomized to high-flow oxygen and in 17 (17.0%) of those randomized to conventional oxygen therapy, while bacteremia was detected in 7 (7.1%) vs 11 (11.0%), respectively. CONCLUSIONS AND RELEVANCE: Among patients with severe COVID-19, use of high-flow oxygen through a nasal cannula significantly decreased need for mechanical ventilation support and time to clinical recovery compared with conventional low-flow oxygen therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04609462.

Performance verification of the Abbott SARS-CoV-2 test for qualitative detection of IgG in Cali, Colombia.

BACKGROUND: Adequate testing is critically important for control of the SARS-CoV-2 pandemic. Antibody testing is an option for case management and epidemiologic studies, with high specificity and variable sensitivity. However, characteristics of local populations may affect performance of these tests. For this reason, the National Institute of Health (INS) and regulatory agencies in Colombia require verification of diagnostic accuracy of tests introduced to the Colombian market. METHODS: We conducted a validation study of the Abbott SARS-CoV-2 test for qualitative detection of IgG using the Abbott Architect i2000SR. Participants and retrospective samples were included from patients with suspected SARS-CoV-2 infection, age ≥18 years, and ≥8 days elapsed since initiation of symptoms. Pre-pandemic plasma samples (taken before October 2019) were used as controls. We estimated the sensitivity, specificity and agreement (kappa) of the Abbott IgG test compared to the gold standard (RT-PCR). RESULTS: The overall sensitivity was 83.1% (95% CI: 75.4-100). Sensitivity among patients with ≥14 days since the start of symptoms was 85.7%, reaching 88% in samples collected from patients with COVID-19 symptoms onset >60 days. Specificity was 100% and the kappa index of agreement was 0.804 (95% CI: 0.642-0.965). CONCLUSIONS: Our findings show high sensitivity and specificity of the Abbott IgG test in a Colombian population, which meet the criteria set by the Colombian INS to aid in the diagnosis of COVID-19. Data from our patient groups also suggest that IgG response is detectable in a high proportion of individuals (88.1%) during the first two months following onset of symptoms.

Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil.

Bats are important for the homeostasis of ecosystems and serve as hosts of various microorganisms including bacteria, viruses, and fungi with pathogenic potential. This study aimed to isolate fungi from biological samples obtained from bats captured in the city of Sinop (state of Mato Grosso, Brazil), where large areas of deforestation exist due to urbanization and agriculture. On the basis of the flow of people and domestic animals, 48 bats were captured in eleven urban forest fragments. The samples were processed and submitted to microbiological cultures, to isolate and to identify the fungal genera. Thirty-four (70.83%) of the captured bats were positive for fungi; 18 (37.5%) and 16 (33.33%) of these bats were female and male, respectively. Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp., and Candida sp., which may cause opportunistic infections, were isolated. The bat species with the highest number of fungal isolates was Molossus molossus: 21 isolates (43.8%). According to our results, bats captured in urban forest fragments in Sinop harbor pathogenic fungi, increasing the risk of opportunistic fungal infections in humans and domestic animals.

Evaluation of sensitization to the crude extract of Dermatophagoides farinae and its derived allergens, Der f 2 and Zen 1, in dogs with atopic dermatitis in Southern Brazil.

BACKGROUND: Atopic dermatitis is associated with the production of IgE antibodies against environmental allergens and allergens of the house dust miteDermatophagoides farinae are frequently implicated in the disease. OBJECTIVES: We aimed to observe the allergen-specific IgE against crudeD. farinae, Der f 2 and Zen 1 in dogs with atopic dermatitis and report if these dogs are in contact with material that could shelter mite allergens. METHODS: 100 dogs with clinical diagnosis of atopic dermatitis were included after exclusion of other forms of pruritic skin disease and dogs that already received specific or non-specific immunotherapy. These dogs were of different breeds and ages and they were presented at a veterinary teaching hospital and a private service of veterinary dermatology, both located in Curitiba, Southern Brazil. At the time of anamnesis, some questions were applied to know the possibility of these dogs having had contact with furniture and textile material which could shelter house dust mites. Sera samples were obtained and further analyzed by ELISA assay to measure serum IgE levels against these allergens with an established cut-off of 0.200 IgE optical density. RESULTS: The allergen-specific IgE positivity against crudeD. farinae (92 %) and Zen 1 (77 %) was higher than Der f 2 (56 %). There was a correlation in sensitization to crude D. farinae and Zen 1 that was not observed between crude D. farinae and Der f 2 and Der f 2 and Zen 1. The sensitization to D. farinae and its allergens was associated with an unrestricted exposition to furniture and textile material. CONCLUSION & CLINICAL RELEVANCE: dogs with atopic dermatitis are frequently sensitized to D. farinae and its allergens, Der f 2 and Zen 1, may be considered major allergens in these dogs. Zen 1 may be the main allergen responsible for the sensitization to crude D. farinae.

Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles With Simple Haloalkanes.

Selective alkylation of pyrazoles could solve a challenge in chemistry and streamline synthesis of important molecules. Here we report catalyst-controlled pyrazole alkylation by a cyclic two-enzyme cascade. In this enzymatic system, a promiscuous enzyme uses haloalkanes as precursors to generate non-natural analogs of the common cosubstrate S-adenosyl-l-methionine. A second engineered enzyme transfers the alkyl group in highly selective C-N bond formations to the pyrazole substrate. The cosubstrate is recycled and only used in catalytic amounts. Key is a computational enzyme-library design tool that converted a promiscuous methyltransferase into a small enzyme family of pyrazole-alkylating enzymes in one round of mutagenesis and screening. With this enzymatic system, pyrazole alkylation (methylation, ethylation, propylation) was achieved with unprecedented regioselectivity (>99 %), regiodivergence, and in a first example on preparative scale.

Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics.

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design.

Botulinum toxin therapy in Frey's syndrome: a retrospective study of 440 treatments in 100 patients.

OBJECTIVE: Frey's syndrome is characterised as sweating, redness and warmth of the parotideal area and is often treated with botulinum toxin A. The objective of this retrospective study was to prove whether the toxin dosage and time-to-treatment intervals change after repeated botulinum toxin injections. STUDY DESIGN/METHODS: The charts of patients, who were treated for Frey's syndrome during the last 16 years, were assessed. Three brands of botulinum toxin A were available for therapy. The Minor test was used to confirm the sweating before each treatment and to determine the toxin dosage. Constant amount of botulinum toxin was injected per cm2 of the affected area. Patients consulted our department for the next treatment as soon as they felt disturbed by recurring sweating and when the sweating was objectively evident in the Minor test. Time intervals between treatments and injected toxin dosages were assessed. RESULTS: In total, 100 patients received 440 treatments in 16 years. Repeated injections, median 4.0, were carried out in 70.5% of patients. Median time interval to the first injection was 2.8 years. Median time interval between treatments was 12.0 months and showed to be steady (anova, P = .49, F = 1.01). CONCLUSION: Duration of effect of botulinum toxin on Frey's syndrome was long-lasting and stable with no significantly different time intervals between treatments. The extent of the sweating area did not vary significantly after repeated treatments and required a constant dose of botulinum toxin A.

Psychologic theories in functional neurologic disorders.

In this chapter we review key psychologic theories that have been mooted as possible explanations for the etiology of functional neurologic symptoms, conversion disorder, and hysteria. We cover Freudian psychoanalysis and later object relations and attachment theories, social theories, illness behavior, classic and operant conditioning, social learning theory, self-regulation theory, cognitive-behavioral theories, and mindfulness. Dissociation and modern cognitive neuroscience theories are covered in other chapters in this series and, although of central importance, are omitted from this chapter. Our aim is an overview with the emphasis on breadth of coverage rather than depth.

Functional coma.

Functional coma - here defined as a prolonged motionless dissociative attack with absent or reduced response to external stimuli - is a relatively rare presentation. In this chapter we examine a wide range of terms used to describe states of unresponsiveness in which psychologic factors are relevant to etiology, such as depressive stupor, catatonia, nonepileptic "pseudostatus," and factitious disorders, and discuss the place of functional or psychogenic coma among these. Historically, diagnosis of functional coma has sometimes been reached after prolonged investigation and exclusion of other diagnoses. However, as is the case with other functional disorders, diagnosis should preferably be made on the basis of positive findings that provide evidence of inconsistency between an apparent comatose state and normal waking nervous system functioning. In our review of physical signs, we find some evidence for the presence of firm resistance to eye opening as reasonably sensitive and specific for functional coma, as well as the eye gaze sign, in which patients tend to look to the ground when turned on to one side. Noxious stimuli such as Harvey's sign (application of high-frequency vibrating tuning fork to the nasal mucosa) can also be helpful, although patients with this disorder are often remarkably unresponsive to usually painful stimuli, particularly as more commonly applied using sternal or nail bed pressure. The use of repeated painful stimuli is therefore not recommended. We also discuss the role of general anesthesia and other physiologic triggers to functional coma.

[74-year-old patient with cystic pancreatic lesions. An endoscopy-based algorithm]. / 74-jähriger Patient mit zystischen Pankreasraumforderungen. Vorschlag für einen endoskopischen Diagnosealgorithmus.

Often, equivocal pancreatic cystic masses in a patient cannot be clearly identified. We report on a 74-year-old patient who consulted us with size-gaining multi-cystic lesions located at the pancreatic head and tail as well as with an increased CA 19-9 level. By using diagnostic methods as ultrasound, radiological images and innovative endoscopic techniques an intraductal papillary mucinous neoplasm (IPMN) was diagnosed. Evaluation of equivocal cystic lesions requires developing of further strategies as well as integration of new concepts: We present a diagnostic algorithm based on endoscopy that enables us to perform an adapted therapy by having a more accurate evaluation and the opportunity to gain samples where unclear lesions are given.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. METHODS: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. RESULTS: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. CONCLUSION: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.