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Introdução: A anemia grave leva a hipóxia tecidual e ativação de mecanismos compensatórios, com aumento da frequência cardíaca (FC) e frequência respiratória (FR). Como em humanos, a transfusão sanguínea com concentrado de hemácias em cães é uma terapia suporte enquanto se busca o diagnóstico ou o tratamento se torne efetivo. Objetivo: O objetivo do presente estudo foi analisar os parâmetros: frequência cardíaca (FC), frequência respiratória (FR), temperatura retal (T°C) e coloração de mucosas (CM) pré e pós-transfusionais em cães com anemia de causas diversas. Material e métodos: Foi realizada análise retrospectiva das fichas de acompanhamento de transfusões de concentrado de hemácias em um Hospital Veterinário no período de julho de 2023 a junho de 2024. As transfusões foram realizadas em cães de várias raças, idades e peso corpóreo. A hemoglobina destes pacientes variou de 1,5 a 5g/dL. Foram analisados os registros de FC, FR, T°C e CM pré-transfusionais (T1) e pós-transfusionais (T2) de 149 transfusões, sendo que foram retirados do estudo quatro transfusões devido a reação transfusional (três reações febril não hemolítica e uma sobrecarga de volume (TACO)), totalizando 145 transfusões avaliadas. Os dados foram submetidos ao teste t pareado ou de Mann Whitney na dependência da avaliação da normalidade dos dados, considerando p < 0,05 como significativo. Resultados: Houve redução significativa na frequência cardíaca e frequência respiratória após as transfusões: FC T1: 134,9 ± 29,6 bpm; FC T2: 104,3 ± 26,1bpm; FR T1: 28 (mín: 8, max: 80) mpm; FR T2: 24 (mín: 8, max: 80) mpm. Não houve diferença na temperatura retal, T1: 37,7ºC (min: 33,4; max: 40,0) e T2: 37,9ºC (min: 33,0; max:39,7). Das 145 transfusões, houve melhora na CM em 97 transfusões (67%); não houve alterações na CM em 41 transfusões (28%); em 7 transfusões (5%) não foi possível avaliar a mucosa por fatores como agressividade dos animais. Discussão: A redução na FC e FR após as transfusões confirma a eficácia do procedimento na diminuição da hipóxia tecidual e redução na ativação de mecanismos compensatórios. A T°C ao longo das transfusões não apresentou alterações significativas, mostrando que a utilização de sangue refrigerado (à 4°C) em cães não ocasiona diminuição da temperatura corporal. A melhora da coloração das mucosas pode sinalizar o aumento do número de hemácias e/ou diminuição da vasoconstrição periférica. Conclusão: Pode-se concluir com os presentes dados que o acompanhamento da transfusão sanguínea com FC, FR, CM e temperatura é importante para detecção precoce de reação transfusional, mas também para avaliação da melhora dos parâmetros clínicos dos pacientes. Portanto, a transfusão de concentrado de hemácias em cães é eficaz para diminuição da hipóxia tecidual demonstrada pelo acompanhamento de parâmetros facilmente obtidos no exame físico.

Introdução: Diversas doenças em cães causam anemias que requer transfusão sanguínea, podendo causar reações transfusionais. Objetivo: O presente estudo avaliou o número e classificou as reações transfusionais ocorridas em cães que receberam concentrado de hemácias durante o período de janeiro/2022 a junho/2024 em um Hospital Veterinário Universitário. Material e métodos: Foram analisadas de forma retrospectiva 260 fichas de acompanhamento de transfusões de concentrado de hemácias em cães. Dois pacientes foram retirados do estudo pois vieram a óbito no início da transfusão por complicações da doença de base, sendo descartada a possibilidade de reação transfusional. Os pacientes incluídos no estudo receberam bolsas de concentrado de hemácias em taxas que correspondem ao seu quadro clínico, idade e doença e foram acompanhados durante todo o procedimento. As transfusões só ocorreram após verificar a compatibilidade do sangue do doador e receptor, para assim evitar ao máximo reações hemolíticas agudas. Parâmetros clínicos como frequência cardíaca e respiratória, ausculta cárdio-respiratória, temperatura, coloração de mucosa, tempo de preenchimento capilar e outras avaliações clínicas, quando necessárias, eram aferidas/avaliadas antes do início, a cada 15 minutos na primeira hora e a cada 30 minutos até o término da transfusão e após a sua finalização. Após coleta dos dados foi realizado análise descritiva utilizando médias para quantificar a porcentagem de reações transfusionais. Resultados: Neste período o Projeto Vida – Medicina Transfusional, realizou 260 transfusões de concentrado de hemácias em cães, sem distinção de idade, raça, sexo e peso corpóreo. Das 258 transfusões incluídas no estudo, 13 cães (5,03%) apresentaram alterações clínicas compatíveis com reação transfusional e o procedimento foi interrompido e o paciente tratado. Nenhum dos animais veio à óbito. As reações transfusionais diagnosticadas foram: 2,32% (6/258) sobrecarga circulatória (TACO), 2,32% (6/258) reação febril não hemolítica por exclusão de outras causas como contaminação bacteriana e reação imunomediada hemolítica aguda e 0,38% (1/258) hipotensão. Discussão: Transfusão sanguínea salva vidas, mas tem riscos. Como acontece em humanos, em animais as reações transfusionais também têm sido diagnosticadas, porém pouco são os estudos sobre sua incidência e não há um sistema próprio para registro destas reações. Um levantamento realizado em 2009 no mesmo Hospital Veterinário Universitário com o uso ainda de sangue total registrou 15% de reações transfusionais em sua maioria reação imunomediada não hemolítica (alérgica), o que após 2009 com a separação dos componentes sanguíneos estas taxas diminuíram e neste presente estudo apenas 5,03% das transfusões apresentaram intercorrência. Outros estudos mostram resultados variáveis dependendo da reação transfusional como: sobrecarga circulatória de 4,7%, reação febril não hemolítica 1,3 a 24,2% e hipotensão 0,9%. Os resultados deste presente estudo demonstram que a incidência de reações transfusionais está com índices melhores do que os apresentados na literatura. Conclusão: O uso e controle de hemocomponentes, testes de compatibilidade pré-transfusionais, o acompanhamento do paciente durante o procedimento e o reconhecimento das reações transfusionais são condutas que diminuem o risco transfusional e aumentam a segurança da transfusão sanguínea em cães.

The aim of this study was to evaluate the cytotoxicity of the prepared Gold Nanoparticles from Triphala Plant extract and of the indigenous mouthwash prepared from these Triphala based gold nanoparticles (AuNP’s) using Artemia salina test. Its objective was to assess whether the prepared mouthwash was safe and biocompatible to use as an alternative to commercial mouthwashes in orthodontic patients to reduce the plaque load around brackets and bands as an adjuvant to tooth brushing. 15 g of brine shrimps was hatched in salt solution after incubating them for 24 – h. Prepared AuNP’s at concentrations 0µL (microliter), 5µL, 10µL, 20µL, 30µL, 50µL were added using a micropipette in one of the microtiter plates and mouthwash prepared from these AuNP’s were added at concentrations 0µL, 10µL, 20µL, 30µL, 40µL, 50µL in another microtiter plate. 10 Hatched nauplii (brine shrimps) were added in each of these microtiter plates and incubated for 24 – h. All nauplii were alive at the end of 24 – h in the microtiter plate containing AuNP’s. At concentrations 0µL, 10µL, 20µL, 30µL nauplii were alive and at 40µL, 50µL concentrations no nauplii were alive at the end of 24 – h in the microtiter plate containing mouthwash. Triphala mediated AuNP’s showed no toxic effect on the brine shrimps. AuNP’s based mouthwash showed excellent compatibility at lower concentrations. Mild toxic effects were elicited at higher concentrations. Hence, AuNP’s could be used to prepare mouthwashes at 20- 30 µL concentrations.

Background: Effective communication is a key feature of optimal doctor-patient relationship. As the postgraduate curriculum lacks standardized teaching and assessment, this study aimed to assess effectiveness of training program on communication skills of postgraduate medical students. Materials and methods: A prospective interventional study was conducted among first year postgraduate medical students from different clinical specialty. Training program included introduction to principles of communication with basics of doctor-patient relationship, administering informed consent, breaking bad news and counselling patients with HIV/cancer. Pre-test and post-test skill assessment by Faculty and postgraduate (PG) self-rating was carried out using KEECC-A Assessment tool for seven core communication competencies. Perception of students on training program was collected via feedback questionnaire. Results: Forty-three postgraduate medical students participated. Statistically significant increase in communication skills scores post training was observed with mean post-test assessment scores of 24.26±2.94, p

An experiment was conducted to study the effects of water stress on physiological parameters associated to drought tolerance in soybean at the Department of Agronomy, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh during January to April, 2015. Four soybean genotypes namely i) BU Soybean 1 ii) Binasoybean 1 iii) Galarsum and iv) BARI Soybean 5 were grown in two watering regimes viz. control (80% of the field capacity) and water stress (50% of the field capacity). Genotypic variability was found in water stress tolerance in soybean. Highest accumulation of leaf proline, sugar and water content and lower accumulation of malondialdehyde were found in Binasoybean 1 compared to other genotypes. Lowest yield reduction was found in Binasoybean 1. Binasoybean 1 showed relatively higher drought tolerance whereas BARI Soybean 5 was found susceptible to yield. It was found that higher water stress tolerance in Binasoybean 1 was associated with better water relations and higher accumulation of sugar and proline and lower accumulation of malondialdehyde content in leaf.

Periodontally Accelerated Osteogenic Orthodontics (PAOO), a patented technique by the Wilcko brothers has become very popular in reducing the treatment time in Orthodontics. A 17-year-old male patient presented to the Department with a Class I skeletal and a Class I dental pattern with bilateral canine impactions. The canine impaction was buccal on the right side (13) and palatal on the left side (23). The initial treatment plan was to disimpact 13 with a piggy back wire and 23 using the K9 spring respectively and it did not involve the PAOO Technique. However, six months into treatment, the patient stopped reporting for his regular appointments and reported after a time interval of one and a half years to continue his treatment. At the time of his return, considerable bone loss around the region of 23 was noticed on radiographs. The PAOO technique was therefore, carried out to augment bone around 23 as well as reduce the time for treatment.

Kernohan's notch is one of the commonly described false localizing signs in clinical neurology. The lesion has been clearly demonstrated by MRI and is shown as hyperintense lesion of the cerebral peduncle on T2-weighted images. The lesion is best demonstrated on coronal imaging. We describe the lesion in a 56-year-old male with chronic subdural hematoma. The neurological sings disappeared after evacuation of the SDH.

Background: Dental plaque may be attributed as a precursor to various oral health problems like dental caries, periodontal disease, halitosis, etc. With an ever-increasing awareness about the adverse effects of chemical formulations, emphasis is now being laid on the usage of herbal ingredients, as they are safer for long-term use in addition to their medicinal benefits., Aim: The present study aims to assess the anti-plaque and anti-inflammatory efficacy of herbal toothpaste compared to synthetic toothpaste among 20-40-year-old patients in Bareilly, Uttar Pradesh., Methodology: In this study, 130 subjects aged between 20 and 40 years with poor oral hygiene status and signs of gingival inflammation corresponding with chronic marginal gingivitis were selected. Subjects in Group 1 were prescribed herbal toothpaste (KUDOS Ayurveda), whereas patients in Group 2 were administered non-herbal toothpaste. Clinical parameters of gingival health specifically gingival index (GI), patient hygiene performance (PHP), and approximate plaque index (API) were recorded at baseline, 14 days, 28 days, and 42 days. Chi-square test, student-independent t-test, and paired t-test were performed to find significant differences in various variables between the two groups at different follow-up visits., Results: At the end of the study, a significant reduction in GI and API scores was obtained, along with increased PHP scores. The p-value was set at 0.05, and the power of the study was set at 0.95. There was a statistically significant reduction (p < 0.05) in GI and API and an improvement in PHP scores at various time intervals in Group 1 as compared to Group 2.  Conclusion: Regular application of herbal extract dentifrices for 42 days provided a significant reduction of dental plaque and improvement in overall gingival health without any adverse effects. This instilled motivation in the patients to uphold proper oral hygiene. Hence, herbal dentifrices could be prescribed as an adjunct to periodontal therapy in the maintenance phase., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Institute of Dental Sciences issued approval IEC/131/2021. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, KK et al.)

Plasmids encoding bla CTX-M genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT). Here we screened the similarity of 47 bla CTX-M -encoding plasmids, from 45 epidemiologically unrelated and disperse ESBL-E. coli strains, isolated during the early phase (2009-2014) of the ESBL pandemic in western Sweden. Using optical DNA mapping (ODM), both similar and rare plasmids were identified. As many as 57% of the plasmids formed five ODM-plasmid groups of at least three similar plasmids per group. The most prevalent type (28%, IncIl, pMLST37) encoded bla CTX-M-15 (n = 10), bla CTX-M-3 (n = 2) or bla CTX-M-55 (n = 1). It was found in isolates of various sequence types (STs), including ST131. This could indicate ongoing local HGT as whole-genome sequencing only revealed similarities with a rarely reported, IncIl plasmid. The second most prevalent type (IncFII/FIA/FIB, F1:A2:B20) harboring bla CTX-M-27 , was detected in ST131-C1-M27 isolates, and was similar to plasmids previously reported for this subclade. The results also highlight the need for local surveillance of plasmids and the importance of temporospatial epidemiological links so that detection of a prevalent plasmid is not overestimated as a potential plasmid transmission event in outbreak investigations., (© 2024. The Author(s).)

Background and Purpose: Chronic pain remains a major clinical problem that needs effective therapeutic agents. Glutamate delta 1 (GluD1) receptors and the protein cerebellin 1 (Cbln1) are down-regulated in the central amygdala (CeA) in models of inflammatory and neuropathic pain. One treatment with Cbln1, intracerebroventricularly (ICV) or in CeA, normalized GluD1 and reduced AMPA receptor expression, resulting in lasting (7-10 days) pain relief. Unlike many CNS-targeting biological agents, the structure of Cbln1 suggests potential blood-brain barrier penetration. Here, we have tested whether systemic administration of Cbln1 provides analgesic effects via action in the CNS., Experimental Approach: Analgesic effects of intravenous recombinant Cbln1 was assessed in complete Freund's adjuvant inflammatory pain model in mice. GluD1 knockout and a mutant form of Cbln1 were used., Key Results: A single intravenous injection of Cbln1 mitigated nocifensive and averse behaviour in both inflammatory and neuropathic pain models. This effect of Cbln1 was dependent on GluD1 receptors and required binding to the amino terminal domain of GluD1. Time course of analgesic effect was similar to previously reported ICV and intra-CeA injection. GluD1 in both spinal cord and CeA was down -regulated in the inflammatory pain model, whereas GluD1 expression in spinal cord but not in CeA, was partly normalized by intravenous Cbln1. Importantly, recombinant Cbln1 was detected in the synaptoneurosomes in spinal cord but not in the CeA., Conclusions and Implications: Our results describe a novel mechanism by which systemic Cbln1 induces analgesia potentially by central actions involving normalization of signalling by spinal cord GluD1 receptors., (© 2023 British Pharmacological Society.)

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data., (© 2024. The Author(s).)

Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently-labeled amyloid fibrils in solution, monitoring the extension and emission intensity of individual fibrils confined in nanochannels with a depth of 300 nm and a width that gradually increases from 300 to 3000 nm. The change in fibril extension with channel width permitted accurate determination of the persistence length of individual fibrils using Odijk's theory for strongly confined polymers. The technique was applied to amyloid fibrils prepared from the Alzheimer's related peptide amyloid-β(1-42) and the Parkinson's related protein α-synuclein, obtaining mean persistence lengths of 5.9 ± 4.5 μm and 3.0 ± 1.6 μm, respectively. The broad distributions of fibril persistence lengths indicate that amyloid fibril polymorphism can manifest in their physical properties. Interestingly, the α-synuclein fibrils had lower persistence lengths than the amyloid-β(1-42) fibrils, despite being thicker. Furthermore, there was no obvious within-sample correlation between the fluorescence emission intensity per unit length of the labelled fibrils and their persistence lengths, suggesting that stiffness may not be proportional to thickness. We foresee that the nanofluidics methodology established here will be a useful tool to study amyloid fibrils on the single fibril level to gain information on heterogeneity in their physical properties and interactions.

Background: Parvalbumin interneuron (PVI) activity synchronizes the medial prefrontal cortex circuit for normal cognitive function, and its impairment may contribute to schizophrenia (SZ). NMDA receptors in PVIs participate in these activities and form the basis for the NMDA receptor hypofunction hypothesis of SZ. However, the role of the GluN2D subunit, which is enriched in PVIs, in regulating molecular networks relevant to SZ is unknown., Methods: Using electrophysiology and a mouse model with conditional deletion of GluN2D from PVIs (PV-GluN2D knockout [KO]), we examined the cell excitability and neurotransmission in the medial prefrontal cortex. Histochemical, RNA sequencing analysis and immunoblotting were conducted to understand molecular mechanisms. Behavioral analysis was conducted to test cognitive function., Results: PVIs in the medial prefrontal cortex were found to express putative GluN1/2B/2D receptors. In a PV-GluN2D KO model, PVIs were hypoexcitable, whereas pyramidal neurons were hyperexcitable. Excitatory neurotransmission was higher in both cell types in PV-GluN2D KO, whereas inhibitory neurotransmission showed contrasting changes, which could be explained by reduced somatostatin interneuron projections and increased PVI projections. Genes associated with GABA (gamma-aminobutyric acid) synthesis, vesicular release, and uptake as well as those involved in formation of inhibitory synapses, specifically GluD1-Cbln4 and Nlgn2, and regulation of dopamine terminals were downregulated in PV-GluN2D KO. SZ susceptibility genes including Disc1, Nrg1, and ErbB4 and their downstream targets were also downregulated. Behaviorally, PV-GluN2D KO mice showed hyperactivity and anxiety behavior and deficits in short-term memory and cognitive flexibility., Conclusions: These findings demonstrate that GluN2D in PVIs serves as a point of convergence of pathways involved in the regulation of GABAergic synapses relevant to SZ., (Copyright © 2023 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Identification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions., Methods: In this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections., Results: We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids., Conclusion: The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management., (© 2023. The Author(s).)

In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures. In this work, we show a simple method for passivation or selective biofunctionalization of silica, without the need for polymerization reactions or vapor-phase deposition. The surface is simply exposed stepwise to three different chemicals over the course of ∼1 h. First, the use of aminopropylsilatrane is used to create a monolayer of amines, yielding more uniform layers than conventional silanization protocols. Second, a cross-linker layer and click chemistry are used to make the surface reactive toward thiols. In the third step, thick and dense poly(ethylene glycol) brushes are prepared by a grafting-to approach. The modified surfaces are shown to be superior to existing options for silica modification, exhibiting ultralow fouling (a few ng/cm 2 ) after exposure to crude serum. In addition, by including a fraction of biotinylated polymer end groups, the surface can be functionalized further. We show that avidin can be detected label-free from a serum solution with a selectivity (compared to nonspecific binding) of more than 98% without the need for a reference channel. Furthermore, we show that our method can passivate the interior of 150 nm × 100 nm nanochannels in silica, showing complete elimination of adsorption of a sticky fluorescent protein. Additionally, our method is shown to be compatible with modifications of solid-state nanopores in 20 nm thin silicon nitride membranes and reduces the noise in the ion current. We consider these findings highly important for the broad field of nanobiotechnology, and we believe that our method will be very useful for a great variety of surface-based sensors and analytical devices.

Objectives: Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1 ., Methods: ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing., Results: We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified bla CTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample., Conclusions: Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

A simple yet efficient assay for the quantitation of proteins ranging from plasma proteins to purified proteins from whole cell lysate, based on the bioconjugation reaction between protein and Meldrum's acid Activated Furan (MAF) is described. This easy to use, sensitive method is based on the conjugation of amine functionalities present on the protein with MAF to form the corresponding Donor Acceptor Stenhouse Adducts (DASAs) with characteristic absorption in the visible region. The reaction is rapid as well as reproducible and shows a proportionate increase in color change over a broad range of protein concentration. The assay was found to be sensitive up to 0.125 mg/mL concentration of the protein and was compatible with most of the commonly employed detergents and isolation protocols which makes it ideal for the estimation of protein samples containing detergents. Another striking feature of this protocol is its tolerance towards other major interference contributors such as chelating agents, reducing agents, carbohydrates and protease inhibitors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

Catalyst activity can depend distinctly on nanoparticle size and shape. Therefore, understanding the structure sensitivity of catalytic reactions is of fundamental and technical importance. Experiments with single-particle resolution, where ensemble-averaging is eliminated, are required to study it. Here, we implement the selective trapping of individual spherical, cubic, and octahedral colloidal Au nanocrystals in 100 parallel nanofluidic channels to determine their activity for fluorescein reduction by sodium borohydride using fluorescence microscopy. As the main result, we identify distinct structure sensitivity of the rate-limiting borohydride oxidation step originating from different edge site abundance on the three particle types, as confirmed by first-principles calculations. This advertises nanofluidic reactors for the study of structure-function correlations in catalysis and identifies nanoparticle shape as a key factor in borohydride-mediated catalytic reactions.

Optical nanoantennas provide control of light at the nanoscale, which makes them important for diverse areas ranging from photocatalysis and flat metaoptics to sensors and biomolecular tweezing. They have traditionally been limited to metallic and dielectric nanostructures that sustain plasmonic and Mie resonances, respectively. More recently, nanostructures of organic J-aggregate excitonic materials have been proposed capable of also supporting nanooptical resonances, although their advance has been hampered from difficulty in nanostructuring. Here, the authors present the realization of organic J-aggregate excitonic nanostructures, using nanocylinder arrays as model system. Extinction spectra show that they can sustain both plasmon-like resonances and dielectric resonances, owing to the material providing negative and large positive permittivity regions at the different sides of its exciton resonance. Furthermore, it is found that the material is highly anisotropic, leading to hyperbolic and elliptic permittivity regions. Nearfield analysis using optical simulation reveals that the nanostructures therefore support hyperbolic localized surface exciton resonances and elliptic Mie resonances, neither of which has been previously demonstrated for this type of material. The anisotropic nanostructures form a new type of optical nanoantennas, which combined with the presented fabrication process opens up for applications such as fully organic excitonic metasurfaces., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Cardiac Failure is an organ system failure with a time sensitive treatment and is usually due to the failure of the left ventricle. Left ventricular function is most easily calculated by a 2D echocardiogram (ECHO). The 2D echo estimates accurately the ejection fraction(EF) of the heart thus, indicating the functioning of the left ventricle. But because of the restrains in availability of the 2D echo in rural set ups of India, it is essential to develop another method to calculate left ventricular function, which is cheap, rapid and accessible in peripheral settings. The objective of this study to calculate the sensitivity and specificity to predict low ejection fraction by Electrocardiogram (ECG)., Material: This cross-sectional study was conducted in medicine department of B R Ambedkar medical college and hospital in Bangalore, from 1st November, 2019 to 31st August, 2021. The consenting participants underwent ECG and 2D ECHO. The ejection fraction values were obtained from the ECG using the formula (2.808 x QRS complex of aVR + 27.198) and by 2D Echo using the Simpson's method. Comparison of ECG and 2D ECHO derived ejection fraction was done, and the sensitivity and specificity was calculated for prediction of low ejection fraction by ECG, followed by stratification for age., Observation: A total of 340 subjects were included in this study. it was found that the EF value calculated using the ECG showed a sensitivity of 87.28% and a specificity of 40.12% to predict EF&lt; 50%, with an accuracy of 64.12%. While the sensitivity and specificity to predict very low EF&lt; 35% was 6.06% and 98.91% respectively, with an accuracy of 80.88%. The sensitivity and specificity of ECG to predict low EF was more for ages above 40 years., Conclusion: The ECG is found to be a good bedside screening tool of the left ventricle ejection fraction in age groups above 40 years of age. In primary care set ups in developing countries like India, the ECG can be used for decisions regarding emergency management and for referral to a cardiac centre/cardiologist, though it is always suggested to be followed up by a 2D- Echocardiogram whenever indicated. It can also be used to avoid unnecessary expensive testing like 2D ECHO, for patients belonging to poorer socio-economic status., (© Journal of the Association of Physicians of India 2011.)

The glutamate delta family of receptors is composed of GluD1 and GluD2 and serve as synaptic organizers. We have previously demonstrated several autism-like molecular and behavioral phenotypes including an increase in dendritic spines in GluD1 knockout mice. Based on previous reports we evaluated whether disruption of autophagy mechanisms may account for these phenotypes. Mouse model with conditional deletion of GluD1 from excitatory neurons in the corticolimbic regions was utilized. GluD1 loss led to overactive Akt-mTOR pathway, higher p62 and a lower LC3-II/LC3-I ratio in the somatosensory cortex suggesting reduced autophagy. Excitatory elements were increased in number but had immature phenotype based on puncta size, lower AMPA subunit GluA1 expression and impaired development switch from predominantly GluN2B to mixed GluN2A/GluN2B subunit expression. Overactive Akt-mTOR signaling and impaired autophagy was also observed in dorsal striatum upon conditional ablation of GluD1 and in the prefrontal cortex and hippocampus in constitutive knockout. Finally, cognitive deficits in novel object recognition test and fear conditioning were observed in mice with conditional ablation of GluD1 from the corticolimbic regions. Together, these results demonstrate a novel function of GluD1 in the regulation of autophagy pathway which may underlie autism phenotypes and is relevant to the genetic association of GluD1 coding, GRID1 gene with autism and other developmental disorders., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pulmonary tuberculosis is one of the common diseases with high prevalence of mortality and morbidity in developing countries. It is one of the rare pulmonary infections which can induce hyponatremia and it is important to recognise hyponatremia because of its potential hazards., Objectives: To assess the serum sodium levels in patients with pulmonary tuberculosis and to establish a relation between the serum sodium levels and its effect on illness in these patients., Material: This is a single-center prospective observational Study conducted on patients with Pulmonary Tuberculosis, irrespective of treatment, in the department of General Medicine, B. R. Ambedkar Medical College, Bangalore from August 2019 to June 2021after obtaining ethical clearance. A total of 100 patients were enrolled in the study who met the predefined inclusion criteria of age more than 18 years and having been diagnosed with active tuberculosis. The collected data was analysed using student's T test and Chi-Square Test and the analysis was done using SPSS software version 24.0., Observation: The mean age was 46.46+/- 15.69, with majority of participants in the age group 46-60 years. Gender wise there was male preponderance in our study with 60%. The average serum sodium concentration in our study was 134.20 +/- 5.59 mmol/l, with 44% prevalence of hyponatremia and 4% SIADH Conclusion: Although mild hyponatremia was seen in more than 50% of patients, we found out that hyponatremia in pulmonary tuberculosis is detected in 44% of our patients with male preponderance. The predominant mechanism of hyponatremia was syndrome of inappropriate anti- diuretic hormone secretion (SIADH), which was present in 65% of cases with hyponatremia. Early detection and treatment of underlying electrolyte abnormality can potentially reduce mortality and morbidity associated with tuberculosis and reduce duration of hospitalization. Further research into the prevalence of potassium, magnesium and chloride abnormalities can add to the lacunae of knowledge., (© Journal of the Association of Physicians of India 2011.)

The relationship between TB and DM has been debated. Experts are concerned about the convergence of TB and diabetes epidemics, particularly in low- to middle-income countries like India and China, which have the world's highest TB burden and fastest rising diabetes prevalence. Diabetes mellitus may be related to the rise in TB cases in India. We discuss TB epidemiology, clinical features, microbiology, imaging, treatment, and prognosis., Material: The data was gathered from out-patients and medical ward patients at B.R. Ambedkar Medical College under NTEP and would be used in this research based on inclusion and exclusion criteria. The patient/attendant gave written informed consent. A comprehensive clinical history, physical examination, and appropriate investigations were performed., Study Design: Prospective, observational study., Sample Size: 50. Study place: Department Of General Medicine And Department Of Chest Medicine., Inclusion Criteria: Age group &gt;18 years. Sputum positive and sputum negative cases. Clinical diagnosed diabetes both naive and on treatment., Exclusion Criteria: Age group &lt; 18 years. Pregnant women. Immunocompromised states (HIV positive, Long-term corticosteroid, chemotherapy, post transplant patients, associated oncological disorders)., Observation: The research group had 60% men and 40% females, a 3:2 male to female ratio. 80% of patients were over 40, and 66% were from low socioeconomic backgrounds. Cough with expectoration (90%) was the most common symptom, followed by weight loss (32%), fever (30%), and hemoptysis (4%). Our study's participants comprised 46% smokers and 10% alcoholics. The group had diabetes for an average of 5.4 years and 58 percent had clubbing and 30% had Pallor. The research group's average fasting and postprandial blood sugars were 220.5 and 302.7 mg/dl. The study's HbA1c averaged 9.3. 50% of individuals had lower zone involvement and 36% had middle lung field TB. In 46% of patients, the right side was affected, 44% the left, and 10% had bilateral TB. Infiltrations (50%) and Cavitary lesions (30%) were the most frequent results, with multiple cavities (64.3%). Patients with greater HbA1c (&gt;8%) had more cavities., Conclusion: All patients with pulmonary tuberculosis should be screened for diabetes mellitus and should be effectively treated for the same. Patients with coexisting pulmonary tuberculosis and diabetes mellitus have atypical presentations. Chest radiographs of such patients show multiple cavitations with predominant lower lobe involvement. Pulmonary Tuberculosis patients who have diabetes tend to have higher sputum positivity rates and delayed sputum conversion if glycemic levels are poorly controlled., (© Journal of the Association of Physicians of India 2011.)

Objective: The aim of the study was to investigate the key factors of balance function in the early subacute phase after stroke., Design: Ninety-four stroke patients were included. Multiple variables were evaluated, including demographic factors, clinical variables (stroke type; lesion site; Mini-Mental State Examination; motor strength of the hip, knee, and ankle joints; Fugl-Meyer Assessment of lower extremity), neurophysiological variables (amplitude ratio of somatosensory evoked potential of the tibial nerves), and laterality index of fractional anisotropy of the corticospinal tract using diffusion tensor imaging. Balance function was measured using the Berg Balance Scale., Results: The Berg Balance Scale score was significantly negatively correlated with age and laterality index of fractional anisotropy and positively correlated with Mini-Mental State Examination; Fugl-Meyer Assessment of lower extremity; motor strength of the affected hip, knee, and ankle joint; and somatosensory evoked potential amplitude ratio (P < 0.05). The abnormal somatosensory evoked potential group and poor integrity of the corticospinal tract group showed significantly decreased Berg Balance Scale scores. In multivariable logistic regression analysis, age, Fugl-Meyer Assessment of lower extremity score, and ankle plantar flexion strength were significantly associated with balance function (odds ratios = 0.919, 1.181, and 15.244, respectively, P < 0.05)., Conclusions: Higher age, severe initial motor impairment, and strength of the affected lower limb muscles, especially the ankle plantar flexor, are strongly associated with poor balance function early after stroke., To Claim Cme Credits: Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME., Cme Objectives: Upon completion of this article, the reader should be able to: (1) Enhance ability to evaluate motor and balance function of stroke patients by learning common assessment tools including clinical tests, neurophysiological and neuroimaging studies; (2) Explain the important factors associated with balance function impairment in early subacute stroke patients; and (3) Enhance planning rehabilitation strategies for improvement of balance function according to recovery stage after stroke., Level: Advanced., Accreditation: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: The carbapenemase-encoding gene bla NDM-1 has been reported in Vietnam during the last 10 years, and bla NDM -producing Enterobacteriaceae are now silently and rapidly spreading. A key factor behind dissemination of bla NDM-1 is plasmids, mobile genetic elements that commonly carry antibiotic resistance genes and spread via conjugation. The diversity of bla NDM-1 -encoding plasmids from neonates at a large Vietnamese hospital was characterized in this study., Methods: 18 fecal Klebsiella pneumoniae and Klebsiella quasipneumoniae isolates collected from 16 neonates at a large pediatric hospital in Vietnam were studied using optical DNA mapping (ODM) and next-generation sequencing (NGS). Plasmids carrying the bla NDM-1 gene were identified by combining ODM with Cas9 restriction. The plasmids in the isolates were compared to investigate whether the same plasmid was present in different patients., Results: Although the same plasmid was found in some isolates, ODM confirmed that there were at least 10 different plasmids encoding bla NDM-1 among the 18 isolates, thus indicating wide plasmid diversity. The ODM results concur with the NGS data. Interestingly, some isolates had two distinct plasmids encoding bla NDM-1 that could be readily identified with ODM. The coexistence of different plasmids carrying the same bla NDM-1 gene in a single isolate has rarely been reported, probably because of limitations in plasmid characterization techniques., Conclusions: The plasmids encoding the bla NDM-1 gene in this study cohort were diverse and may represent a similar picture in Vietnamese society. The study highlights important aspects of the usefulness of ODM for plasmid analysis., Competing Interests: Competing Interests None to declare., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Iron is an essential trace element required for several vital physiological and developmental processes, including erythropoiesis, bone, and neuronal development. Iron metabolism and oxygen homeostasis are interlinked to perform a vital role in the functionality of the heart. The metabolic machinery of the heart utilizes almost 90 % of oxygen through the electron transport chain. To handle this tremendous level of oxygen, the iron metabolism in the heart is utmost crucial. Iron availability to the heart is therefore tightly regulated by (i) the hepcidin/ferroportin axis, which controls dietary iron absorption, storage, and recycling, and (ii) iron regulatory proteins 1 and 2 (IRP1/2) via hypoxia inducible factor 1 (HIF1) pathway. Despite iron being vital to the heart, recent investigations have demonstrated that iron imbalance is a common manifestation in conditions of heart failure (HF), since free iron readily transforms between Fe 2+ and Fe 3+ via the Fenton reaction, leading to reactive oxygen species (ROS) production and oxidative damage. Therefore, to combat iron-mediated oxidative stress, targeting Nrf2/ARE antioxidant signaling is rational. The involvement of Nrf2 in regulating several genes engaged in heme synthesis, iron storage, and iron export is beginning to be uncovered. Consequently, it is possible that Nrf2/hepcidin/ferroportin might act as an epicenter connecting iron metabolism to redox alterations. However, the mechanism bridging the two remains obscure. In this review, we tried to summarize the contemporary insight of how cardiomyocytes regulate intracellular iron levels and discussed the mechanisms linking cardiac dysfunction with iron imbalance. Further, we emphasized the impact of Nrf2 on the interplay between systemic/cardiac iron control in the context of heart disease, particularly in myocardial ischemia and HF., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids-circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae , we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the bla NDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the bla CTX-M-15 and bla OXA-232 genes in the ST16 isolates were encoded on separate plasmids from the bla NDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding bla NDM-1 .

Plasmid-mediated multidrug resistance in E. coli is becoming increasingly prevalent. Considering this global threat to human health, it is important to understand how plasmid-mediated resistance spreads. From a cohort of 123 patients with recurrent urinary tract infections (RUTI) due to extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL E. coli ), only five events with a change of ESBL E. coli strain between RUTI episodes were identified. Their bla CTX-M encoding plasmids were compared within each pair of isolates using optical DNA mapping (ODM) and PCR-based replicon typing. Despite similar bla CTX-M genes and replicon types, ODM detected only one case with identical plasmids in the sequential ESBL E. coli strains, indicating that plasmid transfer could have occurred. For comparison, plasmids from seven patients with the same ESBL E. coli strain reoccurring in both episodes were analyzed. These plasmids (encoding bla CTX-M-3 , bla CTX-M-14 , and bla CTX-M-15 ) were unaltered for up to six months between recurrent infections. Thus, transmission of bla CTX-M plasmids appears to be a rare event during the course of RUTI. Despite the limited number ( n = 23) of plasmids investigated, similar bla CTX-M - 15 plasmids in unrelated isolates from different patients were detected, suggesting that some successful plasmids could be associated with specific strains, or are more easily transmitted.

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We demonstrate how a recently developed nanofluidic device can be used to study protein-induced compaction of genome-length DNA freely suspended in solution. The protein we use in this study is the hepatitis C virus core protein (HCVcp), which is a positively charged, intrinsically disordered protein. Using nanofluidic devices in combination with fluorescence microscopy, we observe that protein-induced compaction preferentially begins at the ends of linear DNA. This observation would be difficult to make with many other single-molecule techniques, which generally require the DNA ends to be anchored to a substrate. We also demonstrate that this protein-induced compaction is reversible and can be dynamically modulated by exposing the confined DNA molecules to solutions containing either HCVcp (to promote compaction) or Proteinase K (to disassemble the compact nucleo-protein complex). Although the natural binding partner for HCVcp is genomic viral RNA, the general biophysical principles governing protein-induced compaction of DNA are likely relevant for a broad range of nucleic acid-binding proteins and their targets., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control., Methods: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria., Results: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles., Conclusions: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Redox homeostasis regulates key cellular signaling in both physiology and pathology. While perturbations result in shifting the redox homeostasis towards oxidative stress are well documented, the influence of reductive stress (RS) in neurodegenerative diseases and its mechanisms are unknown. Here, we postulate that a redox shift towards the reductive arm (through the activation of Nrf2 signaling) will damage neurons and impair neurogenesis. In proliferating and differentiating neuroblastoma (Neuro 2a/N2a) cells, sulforaphane-mediated Nrf2 activation resulted in increased transcription/translation of antioxidants and glutathione (GSH) production along with significantly declined ROS in a dose-dependent manner leading to a reductive-redox state (i.e. RS). Interestingly, this resulted in endoplasmic reticulum (ER) stress leading to subsequent protein aggregation/proteotoxicity in neuroblastoma cells. Under RS, we also observed elevated Tau/α-synuclein and their co-localization with other protein aggregates in these cells. Surprisingly, we noticed that acute RS impaired neurogenesis as evidenced from reduced neurite outgrowth/length. Furthermore, maintaining the cells in a sustained RS condition (for five consecutive generations) dramatically reduced their differentiation and prevented the formation of axons (p < 0.05). This impairment in RS mediated neurogenesis occurs through the alteration of Tau dynamics i.e. RS activates the pathogenic GSK3β/Tau cascade thereby promoting the phosphorylation of Tau leading to proteotoxicity. Of note, intermittent withdrawal of sulforaphane from these cells suppressed the proteotoxic insult and re-activated the differentiation process. Overall, this results suggest that either acute or chronic RS could hamper neurogenesis through GSK3β/TAU signaling and proteotoxicity. Therefore, investigations identifying novel redox mechanisms impacting proteostasis are crucial to preserve neuronal health., (Copyright © 2020. Published by Elsevier B.V.)

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.

Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the bla CTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the bla CTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying bla CTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings. IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals., (Copyright © 2019 Bikkarolla et al.)

From a cohort of 1836 Swedish patients infected with ESBL-producing Enterobacteriaceae (EPE) during 2004-2014, 513 patients with recurrent EPE infection were identified. Only in 14 of the 513 patients was a change of species (ESBL-E. coli to ESBL-K. pneumoniae or vice versa) found between the index and subsequent infection. Eleven sequential urine isolates from 5 of the 14 patients were available for further analysis of possible transfer of ESBL-carrying plasmids. The plasmid content was studied using optical DNA mapping (ODM), PCR-based replicon typing, and ESBL gene sequencing. ODM allowed us to directly compare whole plasmids between isolates and found similar ESBL-carrying plasmids in 3 out of the 5 patients. The ODM results and the rarity in shift of species between ESBL-E. coli and ESBL-K. pneumoniae imply that in recurrent EPE infections interspecies plasmid transfer is uncommon., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Piriformospora indica (Hymenomycetes, Basidiomycota) is an endophytic fungus that colonises plant roots, and was originally isolated from Rajasthan desert. It is comparable to Arbuscular Mycorrhizal (AM) fungi in terms of plant growth promotional effects. P. indica has been used as an ideal example to analyse the mechanisms of mutualistic symbiosis. Major benefit of P. indica over AM fungi is that it is axenically cultivable in different synthetic and complex media. A preliminary attempt was made to scrutinise the role of P. indica co-cultivation on seedling vigour of common vegetables like Cucumis sativus L., Abelmoschus esculentus (L.) Moench, Solanum melongena L. and Capsicum annuum L. The positive effect of P. indica co-culture on seedling performance was compared to the effects of growth hormones like indole acetic acid and benzyl amino purine when supplemented to the MS medium at a concentration of 0.1 mg ml -1 . An exogenous supply of auxin resulted in enhanced production of roots and cytokinin supplement favoured shoot production, whereas P. indica co-culture favoured simultaneous production of shoot and root over the control. P. indica colonisation inside the roots of C. sativus L. was also successfully established. These preliminary results indicate the prospective role of P. indica in vegetable farming through its favourable effect on plant growth.