A perspective of fluorescence microscopy for cellular structural biology with EGFR as witness.

The epidermal growth factor receptor (EGFR) is a poster child for the understanding of receptor behaviour, and of paramount importance to cell function and human health. Cloned almost forty years ago, the interest in EGFR's structure/function relationships remains unabated, not least because changes in oncogenic EGFR mutants are key drivers of the formation of lung and brain tumours. The structure of the assemblies formed by EGFR have been comprehensibly investigated by techniques such as high-resolution X-ray crystallography, NMR and all-atom molecular dynamics (MD) simulations. However, the complexity embedded in the portfolio of EGFR states that are only possible in the physiological environment of cells has often proved refractory to cell-free structural methods. Conversely, some key inroads made by quantitative fluorescence microscopy and super-resolution have depended on exploiting the wealth of structures available. Here, a brief personal perspective is provided on how quantitative fluorescence microscopy and super-resolution methods have cross-fertilised with cell-free-derived EGFR structural information. I primarily discuss areas in which my research group has made a contribution to fill gaps in EGFR's cellular structural biology and towards developing new tools to investigate macromolecular assemblies in cells.

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

A brief history of the octopus imaging facility to celebrate its 10th anniversary.

Octopus (Optics Clustered to OutPut Unique Solutions) celebrated in June 2020 its 10th birthday. Based at Harwell, near Oxford, Octopus is an open access, peer reviewed, national imaging facility that offers successful U.K. applicants supported access to single molecule imaging, confocal microscopy, several flavours of superresolution imaging, light sheet microscopy, optical trapping and cryoscanning electron microscopy. Managed by a multidisciplinary team, Octopus has so far assisted >100 groups of U.K. and international researchers. Cross-fertilisation across fields proved to be a strong propeller of success underpinned by combining access to top-end instrumentation with a strong programme of imaging hardware and software developments. How Octopus was born, and highlights of the multidisciplinary output produced during its 10-year journey are reviewed below, with the aim of celebrating a myriad of collaborations with the U.K. scientific community, and reflecting on their scientific and societal impact.

A single exposure of sediment sulphate-reducing bacteria to oxytetracycline concentrations relevant to aquaculture enduringly disturbed their activity, abundance and community structure.

AIM: Although feed medicated with antibiotics is widely used in animal production to prevent and treat bacterial infections, the effect of these drugs on nontarget anaerobic bacteria is unknown. We aimed to clarify whether a single exposure of sulphate-reducing bacteria (SRB) from a tilapia pond to oxytetracycline (OTC) concentrations relevant to aquaculture impacts their function, abundance and community structure. METHODS AND RESULTS: To demonstrate changes in SO4(2-) content, SRB abundance, dsrB copy number and SRB diversity, sediment mesocosms were spiked with 5, 25, 50 and 75 mg OTC kg(-1) and examined for 30 days by means of ion chromatography, qPCR, cultivation and fluorescent in situ hybridization (FISH). On day 3, we measured higher SO4(2-) concentrations (ca. two-fold) and a reduction in dsrB copy numbers of approximately 50% in the treatments compared to the controls. After 30 days, a subtle yet measurable enrichment of bacteria from the order Desulfovibrionales occurred in mesocosms receiving ≥ 50 mg OTC kg(-1), notwithstanding that SRB counts decreased two orders of magnitude. OTC was dynamically and reversibly converted into 4-epioxytetracycline and other related compounds in a dose-dependent manner during the experiment. CONCLUSIONS: A single exposure to rather high OTC concentrations triggered functional and structural changes in a SRB community that manifested quickly and persisted for a month. SIGNIFICANCE AND IMPACT OF THE STUDY: This study improves our limited knowledge on the ecotoxicology of antibiotics in anaerobic environments.

Study on viability and chondrogenic differentiation of cryopreserved adipose tissue-derived mesenchymal stromal cells for future use in regenerative medicine.

Adipose-derived mesenchymal stromal cells are promising as a regenerative therapy tool for defective tissues in mesenchymal lineage, including fat, bone, cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation is an essential fundamental technology. The aim of this study is to define an adequate protocol for the cryopreservation of adipose-derived mesenchymal stromal cells, by comparing various protocols so as to determine the effects of cryopreservation on viability and chondrogenic differentiation potential of adipose-derived stem cells upon freeze-thawing of AT-MSCs colonies cryopreserved with standard and modified protocols, using flow cytometry and confocal microscopy. The study concludes that adipose-derived mesenchymal stromal cells could be long-term cryopreserved without any loss of their proliferative or differentiation potential.

Assessment of the diagnostic utility of the electroencephalogram in pediatric emergencies.

INTRODUCTION: We analyze the diagnostic utility of urgent EEG (electroencephalogram) performed in children under 16 years of age in our center. MATERIAL AND METHODS: Descriptive, retrospective, observational study of consecutive patients from 0 to 16 years of age, who underwent an urgent EEG for any reason, from January to December 2022. RESULTS: Of the 388 patients, 70 were children: 37 (52.85%) women, and 33 (47.14%) men. Average age: 6.27 ±â€¯4.809. Of the 70 patients, 6 (8.57%) had previous epilepsy. Reasons for consultation: 17 febrile seizures, 10 first focal seizures, 10 first TCG seizures, 6 paroxysmal episodes, 6 absences, 3 myoclonus of extremities, 3 syncope, 2 SE, 2 visual alterations, 2 low level of consciousness, 2 cyanosis, 2 suspected meningitis or encephalitis, 1 choking, 1 atypical headache, 1 chorea, 1 presyncope, 1 language delay. Of the 70 patients, 47 had a normal EEG (67.14%). Of the 47 patients with a normal EEG, 10 were diagnosed with epilepsy, and 3 of them began receiving antiepileptic treatment upon discharge. None of the patients with suspected syncope or paroxysmal disorder (17 patients, 24.28%) had EEG abnormalities. Of the 17 patients with atypical febrile seizures, 3 had EEG abnormalities. CONCLUSIONS: A third of the EEG records performed in the Emergency Department showed alterations, probably due to the time taken. Almost half of the patients with suspected epilepsy or EE showed EEG abnormalities, which confirmed the diagnosis in these cases and encouraged the clinician to start drug treatment. No case with a high suspicion of epilepsy was dismissed due to the normality of the EEG recording in our series. No patient diagnosed with syncope or paroxysmal disorder had EEG abnormalities. Nearly a quarter of patients with atypical febrile seizures showed EEG abnormalities. We barely register cases of status epilepticus, probably due to the degree of complexity of our center.

A highly specific coding system for structural chromosomal alterations.

The Spanish Collaborative Study of Congenital Malformations (ECEMC, from the name in Spanish) has developed a very simple and highly specific coding system for structural chromosomal alterations. Such a coding system would be of value at present due to the dramatic increase in the diagnosis of submicroscopic chromosomal deletions and duplications through molecular techniques. In summary, our new coding system allows the characterization of: (a) the type of structural anomaly; (b) the chromosome affected; (c) if the alteration affects the short or/and the long arm, and (d) if it is a non-pure dicentric, a non-pure isochromosome, or if it affects several chromosomes. We show the distribution of 276 newborn patients with these types of chromosomal alterations using their corresponding codes according to our system. We consider that our approach may be useful not only for other registries, but also for laboratories performing these studies to store their results on case series. Therefore, the aim of this article is to describe this coding system and to offer the opportunity for this coding to be applied by others. Moreover, as this is a SYSTEM, rather than a fixed code, it can be implemented with the necessary modifications to include the specific objectives of each program.

A 'pocket guide' to total internal reflection fluorescence.

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single-molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a 'one stop shop' explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.

[The 2013 Seville Consensus Document on alternatives to allogenic blood transfusion. An update on the Seville Document]. / 2013. Documento Sevilla de Consenso sobre Alternativas a la Transfusión de Sangre Alogénica. Actualización del Documento Sevilla.

Since allogeneic blood transfusion (ABT) is not harmless, multiple alternatives to ABT (AABT) have emerged, though there is great variability in their indications and appropriate use. This variability results from the interaction of a number of factors, including the specialty of the physician, knowledge and preferences, the degree of anemia, transfusion policy, and AABT availability. Since AABTs are not harmless and may not meet cost-effectiveness criteria, such variability is unacceptable. The Spanish Societies of Anesthesiology (SEDAR), Hematology and Hemotherapy (SEHH), Hospital Pharmacy (SEFH), Critical Care Medicine (SEMICYUC), Thrombosis and Hemostasis (SETH) and Blood Transfusion (SETS) have developed a Consensus Document for the proper use of AABTs. A panel of experts convened by these 6 Societies have conducted a systematic review of the medical literature and have developed the 2013 Seville Consensus Document on Alternatives to Allogeneic Blood Transfusion, which only considers those AABT aimed at decreasing the transfusion of packed red cells. AABTs are defined as any pharmacological or non-pharmacological measure aimed at decreasing the transfusion of red blood cell concentrates, while preserving patient safety. For each AABT, the main question formulated, positively or negatively, is: « Does this particular AABT reduce the transfusion rate or not?¼ All the recommendations on the use of AABTs were formulated according to the Grades of Recommendation Assessment, Development and Evaluation (GRADE) methodology.

Subtelomeric deletion of 12p: Description of a third case and review.

Only 12 cases with a cytogenetically visible deletion of the short arm of chromosome 12 (12p) have been reported so far. The difference in clinical features observed in these patients indicates that there is no distinct phenotype associated with this short arm deletion, although the existence of a del(12p) syndrome was previously suggested. Besides those 12 reports, only two patients have been described with a subtelomeric 12p deletion; both present in the same family in which the son showed a mild phenotype of moderate mental retardation and behavioral problems and his carrier mother had no apparent phenotype. In this article, we describe the third known patient with a subtelomeric 12p deletion in a young boy with mental retardation and microcephaly, and review the literature.

Clinical diagnosis of toenail onychomycosis is possible in some patients: cross-sectional diagnostic study and development of a diagnostic rule.

BACKGROUND: Suspected toenail onychomycosis is a frequent problem. Clinical diagnosis has been considered inadequate. OBJECTIVES: To assess the diagnostic accuracy of clinical findings for detecting fungi in toenails, and to develop and validate a clinical diagnostic rule aimed at improving dermatologists' diagnosis of onychomycosis. METHODS: A cross-sectional diagnostic study was performed including a total of 277 patients seen by 12 dermatologists. The gold standard was the presence of dermatophytes on culture or a positive nail plate biopsy. For each sign we described prevalence, sensitivity, specificity, positive and negative predictive values, and likelihood ratios for positive and negative results. We developed a diagnostic clinical rule and validated it in a subsample. RESULTS: Helpful findings to predict the presence of fungi are: previous diagnosis of fungal disease; abnormal plantar desquamation (affecting > 25% of the sole); onychomycosis considered the most probable diagnosis by a dermatologist; and presence of interdigital tinea. When dermatologists considered onychomycosis the most probable diagnosis and plantar desquamation was present (13% of patients), the positive predictive value for presence of fungi was 81%. When both signs were absent (34% of patients), the positive predictive value for absence of fungi was 71%. In other situations, clinical diagnosis might not give enough information to decide on therapy. CONCLUSIONS: In 13% of the patients (a large number in absolute terms), when dermatologists consider onychomycosis the most probable diagnosis and plantar desquamation is present, therapy should be started without any further test, as clinical diagnosis is at least as accurate as laboratory tests. In other situations, an optimal management strategy should be defined.

Carbohydrate restriction favorably alters lipoprotein metabolism in Emirati subjects classified with the metabolic syndrome.

BACKGROUND AND AIMS: Carbohydrate restriction (CR) has been shown to improve dyslipidemias associated with metabolic syndrome (MetS). We evaluated the effects of CR on lipoprotein subfractions and apolipoproteins in Emirati adults classified with the MetS. METHODS AND RESULTS: 39 subjects (15 men/24 women) were randomly allocated to a CR diet [20-25% energy from carbohydrate (CHO)] for 12 wk (CRD group) or a combination treatment consisting of CRD for 6 wk followed by the American Heart Association diet (50-55% CHO, AHA group) for an additional 6 wk. All subjects reduced body weight, LDL cholesterol and triglycerides (P<0.01). At baseline all subjects had low concentrations of medium VLDL and total HDL particles associated with the very low plasma triglycerides and HDL cholesterol in this population. After 12 wk, the large VLDL subfraction was decreased over time for subjects in the CRD group (P<0.01) while these changes were not observed in those subjects who changed to the AHA diet. The number of medium and small LDL particles decreased for all subjects rendering a less atherogenic lipoprotein profile. In agreement with these results, a significant decrease in apolipoprotein (apo) B was observed (P<0.01). The medium HDL subfraction and apo A-II, which can be considered pro-atherogenic, were also decreased over time in the CRD group only. CONCLUSIONS: These results suggest that weight loss favorably affects lipoprotein metabolism and that the CRD had a better effect on atherogenic VLDL and HDL than the low fat diet recommended by AHA.

Efficacy and safety assessment of different dosage of benznidazol for the treatment of Chagas disease in chronic phase in adults (MULTIBENZ study): study protocol for a multicenter randomized Phase II non-inferiority clinical trial.

BACKGROUND: Chagas disease (CD) continues to be a neglected infectious disease with one of the largest burdens globally. Despite the modest cure rates in adult chronic patients and its safety profile, benznidazole (BNZ) is still the drug of choice. Its current recommended dose is based on nonrandomized studies, and efficacy and safety of the optimal dose of BNZ have been scarcely analyzed in clinical trials. METHODS/DESIGN: MULTIBENZ is a phase II, randomized, noninferiority, double-blind, multicenter international clinical trial. A total of 240 patients with Trypanosoma CD in the chronic phase will be recruited in four different countries (Argentina, Brazil, Colombia, and Spain). Patients will be randomized to receive BNZ 150 mg/day for 60 days, 400 mg/day for 15 days, or 300 mg/day for 60 days (comparator arm). The primary outcome is the efficacy of three different BNZ therapeutic schemes in terms of dose and duration. Efficacy will be assessed according to the proportion of patients with sustained parasitic load suppression in peripheral blood measured by polymerase chain reaction. The secondary outcomes are related to pharmacokinetics and drug tolerability. The follow-up will be 12 months from randomization to end of study participation. Recruitment was started in April 2018. CONCLUSION: This is a clinical trial conducted for the assessment of different dose schemes of BNZ compared with the standard treatment regimen for the treatment of CD in the chronic phase. MULTIBENZ may help to clarify which is the most adequate BNZ regimen in terms of efficacy and safety, predicated on sustained parasitic load suppression in peripheral blood. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03191162. Registered on 19 June 2017.

[Respiratory care in neonatal intensive care units. Situation in the year 2005]. / Asistencia respiratoria en las unidades de cuidados intensivos neonatales en España: situación en 2005.

AIM: To learn the characteristic of the neonatal intensive care units (NICUs) that offer neonatal respiratory assistance in Spain. MATERIAL AND METHOD: A structured survey was developed and sent to all Spanish neonatal units to learn about the respiratory care offered in 2005. RESULTS: A total of 96 Units answered the survey, with an estimated representatively of 63%, with a range from 3 to 92%, depending on the geographical area. Level IIIc Units were in the upper range. Answer the survey 26 units type IIb (27%), 16 IIIa (17%), 40 IIIb (42%) and 14 IIIc (14%). The total number of level III NICU beds was 541 (1.2 beds per 1000 livebirths; range, 0.7-1.7). The mean number of beds per NICU was 4.1 in level IIIa Units, 2.8 in those IIIb and 14.6 in type IIIc NICUs. In level III NICUs, the bed per physician ratio was 2.4 and that of beds per registered nurse was 2.8 (2.2 in level IIIc NICUs). There were a total 13,219 admissions, 54% of those needed mechanical ventilation (36% in IIIa and 65% in level IIIc NICUs). Oxygen blenders for resuscitation at birth were available in 42% of level IIIb and IIIc NICUs. NICUs had one neonatal ventilator per bed, and 63% of units had high frequency ventilation available. All units had nasal-CPAP systems, 25% of level IIIa Units, 58% IIIb and 64% of those type IIIc had systems for nasal ventilation. All level IIIc and 93% of level IIIb NICUs were able to provide inhaled nitric oxygen therapy. Four NICUS offered ECMO. CONCLUSIONS: The mean number of NICU beds per 1000 livebirths is within the lower limits of those been recommended, and there were wide variations among different geographical areas. A 54% of those babies admitted to NICUs required mechanical ventilation. The mean number of NICU beds per registered nurse was 2.8. There was an adequate number of neonatal ventilators (one per bed) and 63% were able to provide HFV. All NICUs hand n-CPAP systems.

More Than 25 Years Together: Basis for a Long-Lifespan Kidney Transplant.

Patients with graft survival for 20 years or more are not uncommon; they are called ultralong kidney recipients. It is interesting to know if there are patterns in donors and recipients that could be reproduced. A retrospective cohort with 22 adult patients with a kidney renal transplant performed more than 25 years ago is analyzed. The mean of age of the donors was 24 years (median, 21 years); 82% were men and the cause of death was mainly acute traumatic brain injury. Recipients had a mean age of 34 years (median, 36 years) at the time of transplant; the most common underlying renal disease was glomerular, without evidence of recurrence. A total of 16 patients had compatibility in HLA II (1 in 11 cases; 2 in 5 cases). Only 6 patients have had any episode of acute rejection; 3 of them have developed antibodies class I, but no donor-specific antibodies. In this retrospective cohort, increases in donor age are associated with poor renal function. The mean creatinine is 1.43 mg/dL (range, 0.97-2.14 mg/dL) and mean proteinuria is 653.43 mg/g (range, 55-3722 mg/g). The characteristics common in ultralong kidney recipients are young male donors, a shortage of episodes of rejection, and good HLA compatibility, especially in class II antigens.

A dietary intervention with functional foods reduces metabolic endotoxaemia and attenuates biochemical abnormalities by modifying faecal microbiota in people with type 2 diabetes.

AIM: To study the effects of a functional food-based dietary intervention on faecal microbiota and biochemical parameters in patients with type 2 diabetes (T2D). MATERIALS AND METHODS: This placebo-controlled, randomized, double-blind study included 81 patients with T2D divided into two 3-month treatment groups: one following a reduced-energy diet with a dietary portfolio (DP) comprising high-fibre, polyphenol-rich and vegetable-protein functional foods; the other taking a placebo (P). The primary outcome was the effect of the DP on faecal microbiota. Secondary endpoints were biochemical parameters, lipopolysaccharide, branched-chain amino acids, trimethylamine N-oxide, glycosylated haemoglobin (HbA1c) and free fatty acids (FFAs). RESULTS: Patients with T2D exhibited intestinal dysbiosis characterized by an increase in Prevotella copri. Dietary intervention with functional foods significantly modified faecal microbiota compared with P by increasing alpha diversity and modifying the abundance of specific bacteria, independently of antidiabetic drugs. There was a decrease in P. copri and increases in Faecalibacterium prausnitzii and Akkermansia muciniphila, two bacterial species known to have anti-inflammatory effects. The DP group also exhibited significant reductions in areas under the curve for glucose, total and LDL cholesterol, FFAs, HbA1c (P< 0.05), triglycerides and CRP, and an increase in antioxidant activity (P< 0.01) vs. the P group. CONCLUSION: Long-term adherence to a high-fibre, polyphenol-enriched and vegetable-protein-based diet provides benefits for the composition of faecal microbiota, and may offer potential therapies for improvement of glycaemic control, dyslipidaemia and inflammation.

Simultaneous widefield single molecule orientation and FRET microscopy in cells.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

A small and active ring X chromosome in a female with features of Kabuki syndrome.

A ring X chromosome is found in about 6% of patients with Turner syndrome (TS), often with mosaicism for a 45,X cell line. Patients with this karyotype are reported to have a higher incidence of a more severe phenotype including mental retardation. In fact, some studies have shown a correlation between this severity and the presence or absence of an intact and functional X inactivation center (XIST). However, the phenotype of the individuals with r(X) cannot be entirely defined in terms of their X-inactivation patterns. Nevertheless, a small group of these patients have been described to manifest clinical features reminiscent of the Kabuki syndrome. Here we present a female patient with clinical features resembling Kabuki syndrome and a mos 45,X/46,X,r(X) karyotype. Methylation analyses of polymorphic alleles of the androgen receptor gene showed that both alleles were unmethylated suggesting an active ring chromosome. A specific X chromosome array CGH was performed estimating the size of the ring to be 17 Mb, lacking the XIST gene, and including some genes with possible implications in the phenotype of the patient.

Cyclical hypereosinophilia with skin manifestations and a clonal T cell population.

Hypereosinophilia is a common biological finding in clinical practice, in some cases without an identifiable cause. We describe the case of a 59-year-old woman with recurrent attacks of facial angioedema, fever, pruritic cutaneous nodules, and eosinophilia that reached up to 12.7 x 10(9) cells/L during outbreaks. She had experienced 2 episodes every month for the last 12 years, and the episodes resolved with systemic corticosteroids. Other causes of eosinophilia were ruled out. The patient showed an aberrant T cell population with a CD3-CD4+ TCR- phenotype that accounted for up to 22% of circulating lymphocytes. Analysis of the T-cell receptor (TCR) gene showed evidence of clonal rearrangement. During the episodes, this cell population produced high levels of interleukin-5, which returned to normal levels between the outbreaks. However the aberrant T cell population remained unaffected after the treatment. We suggest that lymphocyte immunophenotyping analysis should be included in the diagnostic workup of patients with hypereosinophilic syndrome, including the variant type of episodic angioedema and eosinophilia (Gleich syndrome).

[Benign paroxysmal positional vertigo: provocation and freeing manoeuvres]. / Vértigo posicional paroxístico benigno: maniobras de provocación y liberación.

The benign paroxystic positional vertigo (BPPV) is defined by brief episodic vertigo attacks and accompanied by a rotary-linear nystagmus, triggered by head position changes and is always produced in that position. The theory that better explains the BPPV is canalithiasis: free-floating particles leave the utricular macula and enter one of the semicircular canals, producing an endolymphatic movement that stimulates the cupula and produces vertigo and nystagmus. The diagnosis is based on a typical clinical history, normal ear and neurological examination and provocation maneuvers, such as the Dix-Hallpike test, reproduce the vertigo attacks. The treatments are the liberatory maneuvers, such as the Epley maneuver which makes the vertigo disappear. We present two cases of vertigo with a compatible clinic history of BPPV, where the Dix-Hallpike maneuver confirmed the diagnosis. The treatment in both cases was the Epley maneuver.